Hybrid suppressor tRNA for vertebrate cells

ABSTRACT

This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in vertebrate cells. The components include orthogonal tRNA&#39;s, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNA&#39;s/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in vertebrate cells are also provided. The present invention provides vertebrate cells with translation components, e.g., pairs of orthogonal aminoacyl-tRNA synthetases (O-RSs) and orthogonal tRNA&#39;s (O-tRNA&#39;s) and individual components thereof, that are used in vertebrate protein biosynthetic machinery to incorporate an unnatural amino acid in a growing polypeptide chain, in a vertebrate cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/440,015 filed on Mar. 4, 2009, which is the national phase entry inthe United States under 35 U.S.C. § 371 of International PatentApplication No. PCT/US2007/019655 filed on Sep. 7, 2007, which claimspriority to and the benefit of U.S. provisional patent application Ser.No. 60/843,092, filed Sep. 8, 2006, the specification and disclosure ofwhich are incorporated herein in their entirety for all purposes.

FIELD OF THE INVENTION

The invention pertains to the field of translation biochemistry invertebrate cells. The invention relates to methods for producing andcompositions of orthogonal tRNA's, orthogonal synthetases and pairsthereof, in vertebrate cells. The invention also relates to compositionsof unnatural amino acids, proteins and methods of producing proteins invertebrate cells that include unnatural amino acids.

BACKGROUND OF THE INVENTION

The genetic code of every known organism, from bacteria to humans,encodes the same twenty common amino acids. Different combinations ofthe same twenty natural amino acids form proteins that carry outvirtually all the complex processes of life, from photosynthesis tosignal transduction and the immune response. In order to study andmodify protein structure and function, scientists have attempted tomanipulate both the genetic code and the amino acid sequence ofproteins. However, it has been difficult to remove the constraintsimposed by the genetic code that limit proteins to twenty geneticallyencoded standard building blocks (with the rare exception ofselenocysteine (see, e.g., A. Bock et al., (1991), MolecularMicrobiology 5:515-20) and pyrrolysine (see, e.g., O. Srinivasan, etal., (2002), Science 296:1459-62).

Some progress has been made to remove these constraints, although thisprogress has been limited and the ability to rationally control proteinstructure and function is still in its infancy. For example, chemistshave developed methods and strategies to synthesize and manipulate thestructures of small molecules (see, e.g., E. J. Corey, & X.-M. Cheng,The Logic of Chemical Synthesis (Wiley-Interscience, New York, 1995)).Total synthesis (see, e.g., B. Merrifield, (1986), Science 232:341-7(1986)), and semi-synthetic methodologies (see, e.g., D. Y. Jackson etal., (1994) Science 266:243-7; and, P. E. Dawson, & S. B. Kent, (2000),Annual Review of Biochemistry 69:923-60), have made it possible tosynthesize peptides and small proteins, but these methodologies havelimited utility with proteins over 10 kilo Daltons (kDa). Mutagenesismethods, though powerful, are restricted to a limited number ofstructural changes. In a number of cases, it has been possible tocompetitively incorporate close structural analogues of common aminoacids throughout proteins. See, e.g., R. Furter, (1998), Protein Science7:419-26; K. Kirshenbaum, et al., (2002), ChemBioChem 3:235-7; and, V.Doring et al., (2001), Science 292:501-4.

In an attempt to expand the ability to manipulate protein structure andfunction, in vitro methods using chemically acylated orthogonal tRNA'swere developed that allowed unnatural amino acids to be selectivelyincorporated in response to a nonsense codon, in vitro (see, e.g., J. A.Ellman, et al., (1992), Sience 255:197-200). Amino acids with novelstructures and physical properties were selectively incorporated intoproteins to study protein folding and stability and biomolecularrecognition and catalysis. See, e.g., D. Mendel, et al., (1995), AnnualReview of Biophsics and Biomolecular Structure 24:435-462; and, V. W.Cornish, et al. (Mar. 31, 1995), Angewandte Chemie-International Editionin English 34:621-633. However, the stoichiometric nature of thisprocess severely limited the amount of protein that could be generated.

Unnatural amino acids have been microinjected into cells. For example,unnatural amino acids were introduced into the nicotinic acetylcholinereceptor in Xenopus oocytes (e.g., M. W. Nowak, et al. (1998), In vivoincorporation of unnatural amino acids into ion channels in Xenopusoocyte expression system, Method Enzymol. 293:504-529) by microinjectionof a chemically misacylated Tetrahymena thermophila tRNA (e.g., M. E.Saks, et al. (1996), An engineered Tetrahymena IRNAGln for in vivoincorporation of unnatural amino acids into proteins by nonsensesuppression, J. Biol. Chem. 271:23169-23175), and the relevant mRNA.This has allowed detailed biophysical studies of the receptor in oocytesby the introduction of amino acids containing side chains with uniquephysical or chemical properties. See, e.g., D. A. Dougherty (2000),Unnatural amino acids as probes of protein structure and function, Curr.Opin. Chem. Biol. 4:645-652. Unfortunately, this methodology is limitedto proteins in cells that can be microinjected, and because the relevanttRNA is chemically acylated in vitro, and cannot be re-acylated, theyields of protein are very low.

To overcome these limitations, new components were added to the proteinbiosynthetic machinery of the prokaryote Escherichia coli (E. coli)(e.g., L. Wang, et al., (2001), Science 292:498-500), which allowedgenetic encoding of unnatural amino acids in vivo. A number of new aminoacids with novel chemical, physical or biological properties, includingphotoaffinity labels and photoisomerizable amino acids, keto aminoacids, and glycosylated amino acids have been incorporated efficientlyand with high fidelity into proteins in E. coli in response to the ambercodon, TAG, using this methodology. See, e.g., J. W. Chin et al.,(2002), Journal of the American Chemical Society 124:9026-9027; J. W.Chin, & P. G. Schultz, (2002), ChemBioChem 11:1135-1137; J. W. Chin, etal., (2002), PNAS United States of America 99:11020-11024: and, L. Wang,& P. G. Schultz, (2002), Chem. Comm., 1-10. However, the translationalmachinery of prokaryotes and eukaryotes are not highly conserved; thus,components of the biosynthetic machinery added to E. coli cannot oftenbe used to site-specifically incorporate unnatural amino acids intoproteins in vertebrate cells. For example, the Methanococcus jannaschiityrosyl-tRNA synthetase/tRNA pair that was used in E. coli is notorthogonal in vertebrate cells. In addition, the transcription of tRNAin eukaryotes, but not in prokaryotes, is carried out by RNA PolymeraseIII and this places restrictions on the primary sequence of the tRNAstructural genes that can be transcribed in vertebrate cells. Moreover,in contrast to prokaryotic cells, tRNA's in vertebrate cells need to beexported from the nucleus, where they are transcribed, to the cytoplasm,to function in translation. Finally, the vertebrate 80S ribosome isdistinct from the 70S prokaryotic ribosome. Thus, there is a need todevelop improved components of the biosynthetic machinery to expand thevertebrate genetic code. This invention fulfills these and other needs,as will be apparent upon review of the following disclosure.

SUMMARY OF THE INVENTION

The invention provides vertebrate cells with translation components,e.g., pairs of orthogonal aminoacyl-tRNA synthetases (O-RSs) andorthogonal tRNA's (O-tRNA's) and individual components thereof, that areused in vertebrate protein biosynthetic machinery to incorporate anunnatural amino acid in a growing polypeptide chain, in a vertebratecell.

Compositions of the invention include a vertebrate cell (e.g., amammalian cell, an avian cell, a fish cell, a reptile cell, an amphibiancell, cells derived from non-mammalian animals, etc.) comprising anorthogonal aminoacyl-tRNA synthetase (O-RS) (e.g., derived from anon-vertebrate organism, such as Escherichia coli, Bacillusstearothermophilus, etc.), where the O-RS preferentially aminoacylatesan orthogonal tRNA (O-tRNA) with at least one unnatural amino acid inthe vertebrate cell. Optionally, two or more OtRNA's can beaminoacylated in a given vertebrate cell. In one aspect, an O-RSaminoacylates an O-tRNA with the unnatural amino acid, e.g., at least40%, at least 45%, at least 50%, at least 60%, at least 75%, at least80%, or even 90% or more as efficiently as does an O-RS having an aminoacid sequence, e.g., as set forth in SEQ ID NO.: 86 or 45. In oneembodiment, an O-RS of the invention aminoacylates the O-tRNA with theunnatural amino acid, e.g., at least 10-fold, at least 20-fold, at least30-fold, etc., more efficiently than the O-RS aminoacylates the O-tRNAwith a natural amino acid.

In one embodiment, the O-RS or a portion thereof is encoded by apolynucleotide sequence as set forth in any one of SEQ ID NO.: 3-35, ora complementary polynucleotide sequence thereof. In another embodiment,the O-RS comprises an amino acid sequence as set forth in any one of SEQID NO.: 36-63, and/or 86, or a conservative variation thereof. In yetanother embodiment, the O-RS comprises an amino acid sequence that is,e.g., at least 90%, at least 95%, at least 98%, at least 99%, or atleast 99.5% or more, identical to that of a naturally occurring tyrosylaminoacyl-tRNA synthetase (TyrRS) and comprises two or more amino acidsfrom groups A-E. Group A includes valine, isoleucine, leucine, glycine,serine, alanine, or threonine at a position corresponding to Tyr37 of anE. coli TyrRS. Group B includes aspartate at a position corresponding toAsn126 of an E. coli TyrRS. Group C includes threonine, serine,arginine, asparagine or glycine at a position corresponding to Asp182 ofan E. coli TyrRS. Group D includes methionine, alanine, valine, ortyrosine at a position corresponding to Phe183 of an E. coli TyrRS; and,group E includes serine, methionine, valine, cysteine, threonine, oralanine at a position corresponding to Leu186 of an E. coli TyrRS.

In another embodiment, the O-RS has one or more improved or enhancedenzymatic properties for the unnatural amino acid as compared to anatural amino acid. For example, the improved or enhanced properties forthe unnatural amino acid as compared to a natural amino acid include anyof e.g., a higher Km, a lower Km, a higher kcat, a lower kcat, a lowerkcat/km, a higher kcat/km, etc.

The vertebrate cell also optionally includes an unnatural amino acid(s).The vertebrate cell optionally includes an orthogonal tRNA (O-tRNA)(e.g., derived from a non-vertebrate organism, such as Escherichia coli,Bacillus stearothermophilus, and/or the like), where the O-tRNArecognizes a selector codon and is preferentially aminoacylated with theunnatural amino acid by the O-RS. In one aspect, the O-tRNA mediates theincorporation of the unnatural amino acid into a protein with, e.g., atleast 45%, at least 50%, at least 60%, at least 75%, at least 80%, atleast 90%, at least 95%, or 99% or the efficiency of a tRNA thatcomprises or is processed in a cell from a polynucleotide sequence asset forth in SEQ ID NO.: 65. In another aspect, the O-tRNA comprises thesequence of SEQ ID NO.:65, and the O-RS comprises a polypeptide sequenceselected from an amino acid sequence set forth in any one of SEQ ID NO.:36-63, and/or 86, and/or a conservative variation thereof.

In another embodiment, the vertebrate cell comprises a nucleic acid thatcomprises a polynucleotide that encodes a polypeptide of interest, wherethe polynucleotide comprises a selector codon that is recognized by theO-tRNA. In one aspect, the yield of the polypeptide of interestcomprising the unnatural amino acid is, e.g., at least 2.5%, at least5%, at least 10%, at least 25%, at least 30%, at least 40%, 50% or more,of that obtained for the naturally occurring polypeptide of interestfrom a cell in which the polynucleotide lacks the selector codon. Inanother aspect, the cell produces the polypeptide of interest in theabsence of the unnatural amino acid, with a yield that is, e.g., lessthan 35%, less than 30%, less than 20%, less than 15%, less than 10%,less than 5%, less than 2.5%, etc., of the yield of the polypeptide inthe presence of the unnatural amino acid.

The invention also provides a vertebrate cell comprising an orthogonalaminoacyl-tRNA synthetase (O-RS), an orthogonal tRNA (O-tRNA), anunnatural amino acid, and a nucleic acid that comprises a polynucleotidethat encodes a polypeptide of interest. The polynucleotide comprises aselector codon that is recognized by the O-tRNA. In addition, the O-RSpreferentially aminoacylates the orthogonal tRNA (O-tRNA) with theunnatural amino acid in the vertebrate cell, and the cell produces thepolypeptide of interest in the absence of the unnatural amino acid, witha yield that is, e.g., less than 30%, less than 20%, less than 15%, lessthan 10%, less than 5%, less than 2.5%, etc., of the yield of thepolypeptide in the presence of the unnatural amino acid.

Compositions that include a vertebrate cell comprising an orthogonaltRNA (O-tRNA) are also a feature of the invention. Typically, the O-tRNAmediates incorporation of an unnatural amino acid into a protein that isencoded by a polynucleotide that comprises a selection codon that isrecognized by the O-tRNA in vivo. In one embodiment, the O-tRNA mediatesthe incorporation of the unnatural amino acid into the protein with,e.g., at least 45%, at least 50%, at least 60%, at least 75%, at least80%, at least 90%, at least 95%, or even 99% or more the efficiency of atRNA that comprises or is processed in a cell from a polynucleotidesequence as set forth in SEQ ID NO.: 65. In another embodiment, theO-tRNA comprises or is processed from a polynucleotide sequence as setforth in SEQ ID NO.: 65, or a conservative variation thereof. In yetanother embodiment, the O-tRNA comprises a recyclable O-tRNA.

In one aspect of the invention, the O-tRNA is post-transcriptionallymodified. The invention also provides a nucleic acid that encodes anO-tRNA in a vertebrate cell, or a complementary polynucleotide thereof.In one embodiment, the nucleic acid comprises an A box and a B box.

The invention also features methods of producing translationalcomponents, e.g., O-RSs or O-tRNA/O-RS pairs (and translationalcomponents produced by these methods). For example, the inventionprovides methods of producing an orthogonal aminoacyl-tRNA synthetase(O-RS) that preferentially aminoacylates an orthogonal tRNA with anunnatural amino acid in a vertebrate cell. The method includes, e.g.,(a) subjecting to positive selection, in the presence of an unnaturalamino acid, a population of vertebrate cells of a first species, wherethe vertebrate cells each comprise: i) a member of a library ofaminoacyl-tRNA synthetases (RSs), ii) an orthogonal tRNA (O-tRNA), iii)a polynucleotide that encodes a positive selection marker, and iv) apolynucleotide that encodes a negative selection marker; where cellsthat survive the positive selection comprise an active RS thataminoacylates the orthogonal tRNA (O-tRNA) in the presence of anunnatural amino acid. The cells that survive the positive selection aresubjected to negative selection in the absence of the unnatural aminoacid to eliminate active RSs that aminoacylate the O-tRNA with a naturalamino acid. This provides the O-RS that preferentially aminoacylates theO-tRNA with the unnatural amino acid.

In certain embodiments, the polynucleotide that encodes the positiveselection marker is operably linked to a response element and the cellsfurther comprise a polynucleotide that: a) encodes a transcriptionalmodulator protein (e.g., a vertebrate transcriptional modulator protein,etc.) that modulates transcription from the response element, and b)comprises at least one selector codon. The incorporation of theunnatural amino acid into the transcriptional modulator protein by theO-tRNA aminoacylated with the unnatural amino acid results intranscription of the positive selection marker. In one embodiment, thetranscriptional modulator protein is a transcriptional activator protein(e.g., GAL4, etc.), and the selector codon is an amber stop codon, e.g.,where the amber stop codon is located in or substantially near a portionof the polynucleotide that encodes a DNA binding domain of thetranscriptional activator protein.

The positive selection marker can be any of a variety of molecules. Inone embodiment, the positive selection marker comprises a nutritionalsupplement for growth and the selection is performed on a medium thatlacks the nutritional supplement. In another embodiment, thepolynucleotide that encodes the positive selection marker is, e.g., anura3, leu2, lys2, lacZ gene, his3 (e.g., where the his3 gene encodes animidazole glycerol phosphate dehydratase, detected by providing3-aminotriazole (3-AT)), and/or the like. In yet another embodiment, thepolynucleotide that encodes the positive selection marker comprises aselector codon.

As with the positive selection marker, the negative selection marker canalso be any of a variety of molecules. In certain embodiments, thepolynucleotide that encodes the negative selection marker is operablylinked to a response element from which transcription is mediated by thetranscriptional modulator protein. The incorporation of a natural aminoacid into the transcriptional modulator protein by the O-tRNAaminoacylated with a natural amino acid results in transcription of thenegative selection marker. In one embodiment, the polynucleotide thatencodes the negative selection marker is, e.g., an ura3 gene and thenegative selection is accomplished on a medium that comprises5-fluroorotic acid (5-FOA). In another embodiment, the medium used fornegative selection comprises a selecting or screening agent that isconverted to a detectable substance by the negative selection marker. Inone aspect of the invention, the detectable substance is a toxicsubstance. In one embodiment, the polynucleotide that encodes thenegative selection marker comprises a selector codon.

In certain embodiments, the positive selection marker and/or thenegative selection marker comprises a polypeptide that fluoresces orcatalyzes a luminescent reaction in the presence of a suitable reactant.In one aspect of the invention, the positive selection marker and/or thenegative selection marker is detected by fluorescence-activated cellsorting (FACS), or by luminescence. In certain embodiments, the positiveselection marker and/or negative selection marker comprises an affinitybased screening marker, or a transcriptional modulator protein. In oneembodiment, the same polynucleotide encodes both the positive selectionmarker and the negative selection marker.

In one embodiment, the polynucleotide that encodes the positiveselection marker and/or negative selection marker of the invention cancomprises at least two selector codons, which each or both can compriseat least two different selector codons or at least two of the sameselector codons.

Additional levels of selection/screening stringency can also be used inthe methods of the invention. In one embodiment, the methods cancomprise, e.g., providing a varying amount of an inactive synthetase instep (a), (b) or both (a) and (b), where the varying amount of theinactive synthetase provides an additional level of selection orscreening stringency. In one embodiment, step (a), (b) or both steps (a)and (b) of the method for producing an O-RS includes varying a selectionor screening stringency, e.g., of the positive and/or negative selectionmarker. The method optionally includes subjecting the O-RS thatpreferentially aminoacylates the O-tRNA with the unnatural amino acid toan additional selection round, e.g., an additional positive selectionround(s), an additional negative selection round(s) or combinations ofboth additional positive and negative selection rounds.

In one embodiment, the selecting/screening comprises one or morepositive or negative selection/screening chosen from, e.g., a change inamino acid permeability, a change in translation efficiency, a change intranslational fidelity, etc. The one or more change is based upon amutation in one or more polynucleotide that encodes a component oforthogonal tRNA-tRNA synthetase pair is used to produce protein.

Typically, the library of RSs (e.g., a library of mutant RSs) comprisesRSs derived from at least one aminoacyl-tRNA synthetase (RS), e.g., froma non-vertebrate organism. In one embodiment, the library of RSs isderived from an inactive RS, e.g., where the inactive RS is generated bymutating an active RS. In another embodiment, the inactive RS comprisesan amino acid binding pocket and one or more amino acids that comprisethe binding pocket are substituted with one or more different aminoacids, e.g., the substituted amino acids are substituted with alanines.

In certain embodiments, the method of producing an O-RS further includesperforming random mutation, site-specific mutation, recombination,chimeric construction, or any combination thereof, on a nucleic acidthat encodes an RS, thereby producing the library of mutant RSs. Incertain embodiments, the method further includes, e.g., (c) isolating anucleic acid that encodes the O-RS; (d) generating from the nucleic acida set of polynucleotides that encode mutated O-RSs (e.g., by randommutagenesis, site-specific mutagenesis, chimeric construction,recombination or any combination thereof); and, (e) repeating steps (a)and/or (b) until a mutated O-RS is obtained that preferentiallyaminoacylates the O-tRNA with the unnatural amino acid. In one aspect ofthe invention, steps (c)-(e) are performed at least two times.

Methods of producing O-tRNA/O-RS pairs are also a feature of theinvention. In one embodiment, the O-RS is obtained as described aboveand the O-tRNA is obtained by subjecting to negative selection apopulation of vertebrate cells of a first species, where the vertebratecells comprise a member of a library of tRNA's, to eliminate cells thatcomprise a member of the library of tRNA's that is aminoacylated by anaminoacyl-tRNA synthetase (RS) that is endogenous to the vertebratecells. This provides a pool of tRNA's that are orthogonal to thevertebrate cell of the first species. In one aspect of the invention,the library of tRNA's comprises tRNA's derived from at least one tRNA,e.g., from a non-vertebrate organism. In another aspect of theinvention, the library of aminoacyl-tRNA synthetases (RSs) comprises RSsderived from at least one aminoacyl-tRNA synthetase (RS), e.g., from anon-vertebrate organism. In yet another aspect of the invention, thelibrary of tRNA's comprises tRNA's derived from at least one tRNA from afirst non-vertebrate organism. The library of aminoacyl-tRNA synthetases(RSs) optionally comprises RSs derived from at least one aminoacyl-tRNAsynthetase (RS) from a second non-vertebrate organism. In oneembodiment, the first and second non-vertebrate organisms are the same.Alternatively, the first and second non-vertebrate organisms can bedifferent. Specific O-tRNA/O-RS pairs produced by the methods of theinvention are also a feature of the invention.

Another feature of the invention is a method for producing translationalcomponents in one species and introducing the selected/screenedtranslational components into a second species. For example, the methodof producing a O-tRNA/O-RS pair in a first species (e.g., a vertebratespecies, such as a yeast and the like) further includes introducing anucleic acid that encodes the O-tRNA and a nucleic acid that encodes theO-RS into a vertebrate cell of a second species (e.g., a mammal, aninsect, a fungus, an algae, a plant and the like). The second speciescan use the introduced translational components to incorporate anunnatural amino acid into a growing polypeptide chain in vivo, e.g.,during translation.

In another example, a method of producing an orthogonal aminoacyl-tRNAsynthetase (O-RS) that preferentially aminoacylates an orthogonal tRNAwith an unnatural amino acid in a vertebrate cell includes: (a)subjecting to positive selection, in the presence of an unnatural aminoacid, a population of vertebrate cells of a first species (e.g., avertebrate species, such as a yeast or the like). The vertebrate cellsof the first species each comprise: i) a member of a library ofaminoacyl-tRNA synthetases (RSs), ii) an orthogonal tRNA (O-tRNA), iii)a polynucleotide that encodes a positive selection marker, and iv) apolynucleotide that encodes a negative selection marker. The cells thatsurvive the positive selection comprise an active RS that aminoacylatesthe orthogonal tRNA (O-tRNA) in the presence of an unnatural amino acid.The cells that survive the positive selection are subjected to negativeselection in the absence of the unnatural amino acid to eliminate activeRSs that aminoacylate the O-tRNA with a natural amino acid, therebyproviding an O-RS that preferentially aminoacylates the O-tRNA with theunnatural amino acid. A nucleic acid that encodes the O-tRNA and anucleic acid that encodes the O-RS are introduced into a vertebrate cellof a second species (e.g., mammal, an insect, a fungus, an algae, aplant and/or the like). These components, when translated in the secondspecies, can be used to incorporate unnatural amino acids into a proteinor polypeptide of interest in the second species. In one embodiment, theO-tRNA and/or the O-RS are introduced into a vertebrate cell of a secondspecies.

In certain embodiments, the O-tRNA is obtained by subjecting to negativeselection a population of vertebrate cells of a first species, where thevertebrate cells comprise a member of a library of tRNA's, to eliminatecells that comprise a member of the library of tRNA's that isaminoacylated by an aminoacyl-tRNA synthetase (RS) that is endogenous tothe vertebrate cells. This provides a pool of tRNA's that are orthogonalto the vertebrate cell of the first species and the second species.

Proteins (or polypeptides of interest) with at least one unnatural aminoacid are also a feature of the invention. In certain embodiments of theinvention, a protein with at least one unnatural amino acid includes atleast one post-translational modification. In one embodiment, the atleast one post-translational modification comprises attachment of amolecule (e.g., a dye, a polymer, e.g., a derivative of polyethyleneglycol, a photocrosslinker, a cytotoxic compound, an affinity label, aderivative of biotin, a resin, a second protein or polypeptide, a metalchelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide(e.g., DNA, RNA, etc.), etc.) comprising a second reactive group by a[3+2] cycloaddition to the at least one unnatural amino acid comprisinga first reactive group. For example, the first reactive group is analkynyl moiety (e.g., in the unnatural amino acidp-propargyloxyphenylalanine) (this group is also sometimes refer to asan acetylene moiety) and the second reactive group is an azido moiety.In another example, the first reactive group is the azido moiety (e.g.,in the unnatural amino acid p-azido-L-phenylalanine) and the secondreactive group is the alkynyl moiety. In certain embodiments, a proteinof the invention includes at least one unnatural amino acid (e.g., aketo unnatural amino acid) comprising at least one post-translationalmodification, where the at least one post-translational modificationcomprises a saccharide moiety. In certain embodiments, thepost-translational modification is made in vivo in a vertebrate cell.

In certain embodiments, the protein includes at least onepost-translational modification that is made in vivo by a vertebratecell, where the post-translational modification is not made by aprokaryotic cell. Examples of post-translational modifications include,but are not limited to, acetylation, acylation, lipid-modification,palmitoylation, palmitate addition, phosphorylation, glycolipid-linkagemodification, and the like. In one embodiment, the post-translationalmodification comprises attachment of an oligosaccharide to an asparagineby a GlcNAc-asparagine linkage (e.g., where the oligosaccharidecomprises (GlcNAc-Man)₂-Man-GlcNAc-GlcNAc, and the like). In anotherembodiment, the post-translational modification comprises attachment ofan oligosaccharide (e.g., Gal-GalNAc, Gal-GlcNAc, etc.) to a serine orthreonine by a GalNAc-serine, a GalNAc-threonine, a GlcNAc-serine, or aGlcNAc-threonine linkage. In certain embodiments, a protein orpolypeptide of the invention can comprise a secretion or localizationsequence, an epitope tag, a FLAG tag, a polyhistidine tag, a GST fusion,and/or the like.

Typically, the proteins are, e.g., at least 60%, at least 70%, at least75%, at least 80%, at least 90%, at least 95%, or even at least 99% ormore identical to any available protein (e.g., a therapeutic protein, adiagnostic protein, an industrial enzyme, or portion thereof, and/or thelike), and they comprise one or more unnatural amino acid. In oneembodiment, a composition of the invention includes a protein orpolypeptide of interest and an excipient (e.g., a buffer, apharmaceutically acceptable excipient, etc.).

The protein or polypeptide of interest can contain at least one, atleast two, at least three, at least four, at least five, at least six,at least seven, at least eight, at least nine, or ten or more unnaturalamino acids. The unnatural amino acids can be the same or different,e.g., there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different sitesin the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moredifferent unnatural amino acids. In certain embodiments, at least one,but fewer than all, of a particular amino acid present in a naturallyoccurring version of the protein is substituted with an unnatural aminoacid.

Examples of a protein (or polypeptide of interest) include, but are notlimited to, e.g., a cytokine, a growth factor, a growth factor receptor,an interferon, an interleukin, an inflammatory molecule, an oncogeneproduct, a peptide hormone, a signal transduction molecule, a steroidhormone receptor, erythropoietin (EPO), insulin, human growth hormone,an Alpha-1 antitrypsin, an Angiostatin, an Antihemolytic factor, anantibody, an Apolipoprotein, an Apoprotein, an Atrial natriureticfactor, an Atrial natriuretic polypeptide, an Atrial peptide, a C-X-Cchemokine, T39765, NAP-2, ENA-78, a Gro-a, a Gro-b, a Gro-c, an IP-10, aGCP-2, an NAP-4, an SDF-1, a PF4, a MIG, a Calcitonin, a c-kit ligand, aCC chemokine, a Monocyte chemoattractant protein-1, a Monocytechemoattractant protein-2, a Monocyte chemoattractant protein-3, aMonocyte inflammatory protein-1 alpha, a Monocyte inflammatory protein-1beta, RANTES, I309, R83915, R91733, HCC1, T58847, D31065, T64262, aCD40, a CD40 ligand, a C-kit Ligand, a Collagen, a Colony stimulatingfactor (CSF), a Complement factor 5a, a Complement inhibitor, aComplement receptor 1, DHFR, an epithelial Neutrophil ActivatingPeptide-78, a GROα/MGSA, a GROβ, a GROγ a MIP-1α, a MIP-1δ, a MCP-1, anEpidermal Growth Factor (EGF), an epithelial Neutrophil ActivatingPeptide, an Exfoliating toxin, a Factor IX, a Factor VII, a Factor VIII,a Factor X, a Fibroblast Growth Factor (FGF), a Fibrinogen, aFibronectin, a G-CSF, a OM-CSF, a Glucocerebrosidase, a Gonadotropin, aHedgehog protein, a Hemoglobin, a Hepatocyte Growth Factor (HGF), aHirudin, a Human serum albumin, an ICAM-1, an ICAM-1 receptor, an LFA-1,an LFA-1 receptor, an Insulin-like Growth Factor (IGF), an IGF-I, anIGF-II, an IFN-α, an IFN-β, an IFN-γ, an IL-1, an IL-2, an IL-3, anIL-4, an IL-5, an IL-6, an IL-7, an IL-8, an IL-9, an IL-10, an IL-11,an IL-12, a Keratinocyte Growth Factor (KGF), a Lactoferrin, a leukemiainhibitory factor, a Luciferase, a Neurturin, a Neutrophil inhibitoryfactor (NIF), an oncostatin M, an Osteogenic protein, a Parathyroidhormone, a PD-ECSF, a PDGF, a Pleiotropin, a Protein A, a Protein G, aPyrogenic exotoxins A, B, or C, a Relaxin, a Renin, an SCF, a Solublecomplement receptor I, a Soluble I-CAM 1, a Soluble interleukinreceptors, a Soluble TNF receptor, a Somatomedin, a Somatostatin, aSomatotropin, a Streptokinase, a Superantigens, a Staphylococcalenterotoxins, an SEA, an SEB, an SEC1, an SEC2, an SEC3, an SED, an SEE,a Superoxide dismutase (SOD), a Toxic shock syndrome toxin, a Thymosinalpha 1, a Tissue plasminogen activator, a tumor growth factor (TGF), aTGF-α, a TGF-β, a Tumor Necrosis Factor, a Tumor Necrosis Factor alpha,a Tumor necrosis factor beta, a Tumor necrosis factor receptor (TNFR), aVLA-4 protein, a VCAM-1 protein, a Vascular Endothelial Growth Factor(VEGEF), a Urokinase, a Mos, a Ras, a Raf, a Met; a p53, a Tat, a Fos, aMyc, a Jun, a Myb, a Rel, an estrogen receptor, a progesterone receptor,a testosterone receptor, an aldosterone receptor, an LDL receptor, aSCF/c-Kit, a CD40L/CD40, a VLA-4/VCAM-1, an ICAM-1/LFA-1, ahyalurin/CD44, a corticosterone, a protein present in Genebank or otheravailable databases, and the like, and/or a portion thereof. In oneembodiment, the polypeptide of interest includes a transcriptionalmodulator protein (e.g., a transcriptional activator protein (such asGAL4), or a transcriptional repressor protein, etc.) or a portionthereof.

A vertebrate cell of the invention provides the ability to synthesizeproteins that comprise unnatural amino acids in large useful quantities.For example, proteins comprising an unnatural amino acid can be producedat a concentration of, e.g., at least 10 μg/liter, at least 50 μg/liter,at least 75 μg/liter, at least 100 μg/liter, at least 200 μg/liter, atleast 250 μg/liter, or at least 500 μg/liter or more of protein in acell extract, a buffer, a pharmaceutically acceptable excipient, and/orthe like. In certain embodiments, a composition of the inventionincludes, e.g., at least 10 μg, at least 50 μg, at least 75 μg, at least100 μg, at least 200 μg, at least 250 μg, or at least 500 μg or more ofprotein that comprises a unnatural amino acid.

In certain embodiments, the protein or polypeptide of interest (orportion thereof) is encoded by a nucleic acid. Typically, the nucleicacid comprises at least one selector codon, at least two selectorcodons, at least three selector codons, at least four selector codons,at least five selector codons, at least six selector codons, at leastseven selector codons, at least eight selector codons, at least nineselector codons, or even ten or more selector codons.

The invention also provides methods for producing, in a vertebrate cell,at least one protein comprising at least one unnatural amino acid (aswell as proteins produced by such methods). The methods include, e.g.,growing, in an appropriate medium, a vertebrate cell that comprises anucleic acid that comprises at least one selector codon and encodes theprotein. The vertebrate cell also comprises an orthogonal tRNA (O-tRNA)that functions in the cell and recognizes the selector codon and anorthogonal aminoacyl tRNA synthetase (O-RS) that preferentiallyaminoacylates the O-tRNA with the unnatural amino acid, and the mediumcomprises an unnatural amino acid. In one embodiment, the O-RSaminoacylates the O-tRNA with the unnatural amino acid e.g., at least45%, at least 50%, at least 60%, at least 75%, at least 80%, at least90%, at least 95%, or even 99a % or more as efficiently as does an O-RShaving an amino acid sequence, e.g., as set forth in SEQ ID NO.: 86 or45. In another embodiment, the O-tRNA comprises, is processed from, oris encoded by SEQ ID NO.: 64 or 65, or a complementary polynucleotidesequence thereof. In yet another embodiment, the O-RS comprises an aminoacid sequence as set forth in any one of SEQ ID NO.: 36-63, and/or 86.

In one embodiment, the method further includes incorporating into theprotein the unnatural amino acid, where the unnatural amino acidcomprises a first reactive group; and contacting the protein with amolecule (e.g., a dye, a polymer, e.g., a derivative of polyethyleneglycol, a photocrosslinker, a cytotoxic compound, an affinity label, aderivative of biotin, a resin, a second protein or polypeptide, a metalchelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide(e.g., DNA, RNA, etc.), etc.) that comprises a second reactive group.The first reactive group reacts with the second reactive group to attachthe molecule to the unnatural amino acid through a [3+2] cycloaddition.In one embodiment, the first reactive group is an alkynyl or azidomoiety and the second reactive group is an azido or alkynyl moiety. Forexample, the first reactive group is the alkynyl moiety (e.g., inunnatural amino acid p-propargyloxyphenylalanine) and the secondreactive group is the azido moiety. In another example, the firstreactive group is the azido moiety (e.g., in the unnatural amino acidp-azido-L-phenylalanine) and the second reactive group is the alkynylmoiety.

In certain embodiments, the encoded protein comprises a therapeuticprotein, a diagnostic protein, an industrial enzyme, or portion thereof.In one embodiment, the protein that is produced by the method is furthermodified through the unnatural amino acid. For example, the unnaturalamino acid is modified through, e.g., a nucleophilic-electrophilicreaction, through a [3+2] cycloaddition, etc. In another embodiment, theprotein produced by the method is modified by at least onepost-translational modification (e.g., N-glycosylation, O-glycosylation,acetylation, acylation, lipid-modification, palmitoylation, palmitateaddition, phosphorylation, glycolipid-linkage modification, and thelike) in vivo.

Methods of producing a screening or selecting transcriptional modulatorprotein are also provided (as are screening or selecting transcriptionalmodulator proteins produced by such methods). The methods include, e.g.,selecting a first polynucleotide sequence, where the polynucleotidesequence encodes a nucleic acid binding domain; and mutating the firstpolynucleotide sequence to include at least one selector codon. Thisprovides a screening or selecting polynucleotide sequence. The methodsalso include, e.g., selecting a second polynucleotide sequence, wherethe second polynucleotide sequence encodes a transcriptional activationdomain; providing a construct that comprises the screening or selectingpolynucleotide sequence operably linked to the second polynucleotidesequence; and, introducing the construct, an unnatural amino acid, anorthogonal tRNA synthetase (O-RS) and an orthogonal tRNA (O-tRNA), intoa cell. With these components, the O-RS preferentially aminoacylates theO-tRNA with the unnatural amino acid and the O-tRNA recognizes theselector codon and incorporates the unnatural amino acid into thenucleic acid binding domain, in response to the selector codon in thescreening or selecting polynucleotide sequence. This provides thescreening or selecting transcriptional modulator protein.

In certain embodiments, the compositions and the methods of theinvention include vertebrate cells. A vertebrate cell of the inventionincludes any of, e.g., a mammalian cell, a yeast cell, a fungus cell, aplant cell, an insect cell, etc. The translation components of theinvention can be derived from a variety of organisms, e.g.,non-vertebrate organisms, such as a prokaryotic organism (e.g., E. coli,Bacillus stearothermophilus, or the like), or an archaebacterium, ore.g., a vertebrate organism.

A selector codon of the invention expands the genetic codon framework ofvertebrate protein biosynthetic machinery. Any of a variety of selectorcodons can be used in the invention, including stop codons (e.g., anamber codon, an ochre codon, or an opal stop codon), nonsense codons,rare codons, four (or more) base codons, and/or the like.

Examples of unnatural amino acids that can be used in the compositionsand methods described herein include (but are not limited to): ap-acetyl-L-phenylalanine, a p-iodo-L-phenylalanine, anO-methyl-L-tyrosine, a p-propargyloxyphenylalanine, ap-propargyl-phenylalanine, an L-3-(2-naphthyl)alanine, a3-methyl-phenylalanine, an O-4-allyl- L-tyrosine, a 4-propyl-L-tyrosine,a tri-O-acetyl-GlcNAcβ-serine, an L-Dopa, a fluorinated phenylalanine,an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, a p-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine, aphosphonoserine, a phosphonotyrosine, a p-bromophenylalanine, ap-amino-L-phenylalanine, an isopropyl-L-phenylalanine, an unnaturalanalogue of a tyrosine amino acid; an unnatural analogue of a glutamineamino acid; an unnatural analogue of a phenylalanine amino acid; anunnatural analogue of a serine amino acid; an unnatural analogue of athreonine amino acid; an alkyl, aryl, acyl, azido, cyano, halo,hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl,seleno, ester, thioacid, borate, boronate, phospho, phosphono,phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, oramino substituted amino acid, or any combination thereof; an amino acidwith a photoactivatable cross-linker, a spin-labeled amino acid; afluorescent amino acid; a metal binding amino acid; a metal-containingamino acid; a radioactive amino acid; a photocaged and/orphotoisomerizable amino acid; a biotin or biotin-analogue containingamino acid; a keto containing amino acid; an amino acid comprisingpolyethylene glycol or polyether, a heavy atom substituted amino acid; achemically cleavable or photocleavable amino acid; an amino acid with anelongated side chain; an amino acid containing a toxic group; a sugarsubstituted amino acid; a carbon-linked sugar-containing amino acid; aredox-active amino acid; an α-hydroxy containing acid; an amino thioacid; an α,α disubstituted amino acid; a β-amino acid; a cyclic aminoacid other than proline or histidine, an aromatic amino acid other thanphenylalanine, tyrosine or tryptophan, and/or the like.

The invention also provides polypeptides (O-RSs) and polynucleotides,e.g., O-tRNA's, polynucleotides that encode O-RSs or portions thereof(e.g., the active site of the synthetase), oligonucleotides used toconstruct aminoacyl-tRNA synthetase mutants, polynucleotides that encodea protein or polypeptide of interest that comprise one or more selectorcodon, etc. For example, a polypeptide of the invention includes apolypeptide that comprises an amino acid sequence as set forth in anyone of SEQ ID NO.: 36-63, and/or 86, a polypeptide that comprises anamino acid sequence encoded by a polynucleotide sequence as set forth inany one of SEQ ID NO.: 3-35, and a polypeptide that is specificallyimmunoreactive with an antibody specific for a polypeptide thatcomprises an amino acid sequence as shown in any one of SEQ ID NO.:36-63, and/or 86, or a polypeptide that comprises an amino acid sequenceencoded by a polynucleotide sequence as shown in any one of SEQ ID NO.:3-35.

Also included among the polypeptides of the invention is a polypeptidethat comprises an amino acid sequence that is at least 90% identical tothat of a naturally occurring tyrosyl aminoacyl-tRNA synthetase (TyrRS)(e.g., SEQ ID NO.:2) and comprises two or more amino acids of groups A-E(noted above). Similarly, polypeptides of the invention also optionallyinclude a polypeptide that comprises at least 20 contiguous amino acidsof any one of SEQ ID NO.: 36-63, and/or 86, and two or more amino acidsubstitutions as indicated above in groups A-E. An amino acid sequencecomprising a conservative variation of any of the above polypeptides isalso included as a polypeptide of the invention.

In one embodiment, a composition includes a polypeptide of the inventionand an excipient (e.g., buffer, water, pharmaceutically acceptableexcipient, etc.). The invention also provides an antibody or antiseraspecifically immunoreactive with a polypeptide of the invention.

Polynucleotides are also provided in the invention. Polynucleotides ofthe invention include those that encode proteins or polypeptides ofinterests of the invention with one or more selector codon. In addition,polynucleotides of the invention include, e.g., a polynucleotidecomprising a nucleotide sequence as set forth in any one of SEQ ID NO.:3-35, 64-85; a polynucleotide that is complementary to or that encodes apolynucleotide sequence thereof; and/or a polynucleotide encoding apolypeptide that comprises an amino acid sequence as set forth in anyone of SEQ ID NO.: 36-63, and/or 86, or a conservative variationthereof. A polynucleotide of the invention also includes apolynucleotide that encodes a polypeptide of the invention. Similarly, anucleic acid that hybridizes to a polynucleotide indicated above underhighly stringent conditions over substantially the entire length of thenucleic acid is a polynucleotide of the invention.

A polynucleotide of the invention also includes a polynucleotide thatencodes a polypeptide that comprises an amino acid sequence that is atleast 90% identical to that of a naturally occurring tyrosylaminoacyl-tRNA synthetase (TyrRS) (e.g., SEQ ID NO.: 2) and comprisestwo or more mutations as indicated above in groups A-E (noted above). Apolynucleotide that is that is at least 70%, (or at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or least99% or more) identical to a polynucleotide indicated above and/or apolynucleotide comprising a conservative variation of any of thepolynucleotides indicated above are also included among thepolynucleotides of the invention.

In certain embodiments, a vector (e.g., a plasmid, a cosmid, a phage, avirus, etc.) comprises a polynucleotide of the invention. In oneembodiment, the vector is an expression vector. In another embodiment,the expression vector includes a promoter operably linked to one or moreof the polynucleotides of the invention. In another embodiment, a cellcomprises a vector that includes a polynucleotide of the invention.

In another aspect, the invention provides compositions of compounds andmethods of producing such compounds. For example, compounds include,e.g., an unnatural amino acid (such as p-(propargyloxy)-phenyalanine(e.g., 1 in FIG. 11), azido dyes (such as shown in chemical structure 4and chemical structure 6), an alkynyl polyethylene glycol (e.g., asshown in chemical structure 7), where n is an integer between, e.g., 50and 10,000, 75 and 5,000, 100 and 2,000, 100 and 1,000, etc., and thelike. In embodiment of the invention, the alkynyl polyethylene glycolhas a molecular weight of, e.g., about 5,000 to about 100,000 Da, about20,000 to about 50,000 Da, about 20,000 to about 10,000 Da (e.g., 20,000Da).

Various compositions comprising these compounds, e.g., with proteins andcells, are also provided. In one aspect, the composition that includesthe p-(propargyloxy)-phenyalanine unnatural amino acid, further includesan orthogonal tRNA. The unnatural amino acid can be bonded (e.g.,covalently) to the orthogonal tRNA, e.g., covalently bonded to theorthogonal tRNA though an amino-acyl bond, covalently bonded to a 3′OHor a 2′OH of a terminal ribose sugar of the orthogonal tRNA, etc.

Kits are also a feature of the invention. For example, a kit forproducing a protein that comprises at least one unnatural amino acid ina cell is provided, where the kit includes a container containing apolynucleotide sequence encoding an O-tRNA or an O-tRNA, and apolynucleotide sequence encoding an O-RS or an O-RS. In one embodiment,the kit further includes at least one unnatural amino acid. In anotherembodiment, the kit further comprises instructional materials forproducing the protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows increased expression of hGH using the hybrid tRNA.

DETAILED DESCRIPTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular devices orbiological systems, which can, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. As used in this specification and the appended claims, thesingular forms “a”, “an” and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “acell” includes a combination of two or more cells; reference to“bacteria” includes mixtures of bacteria, and the like.

Unless otherwise defined herein or below in the remainder of thespecification, all technical and scientific terms used herein have thesame meaning as commonly understood by those of ordinary skill in theart to which the invention belongs.

Homologous: Proteins and/or protein sequences are “homologous” when theyare derived, naturally or artificially, from a common ancestral proteinor protein sequence. Similarly, nucleic acids and/or nucleic acidsequences are homologous when they are derived, naturally orartificially, from a common ancestral nucleic acid or nucleic acidsequence. For example, any naturally occurring nucleic acid can bemodified by any available mutagenesis method to include one or moreselector codon. When expressed, this mutagenized nucleic acid encodes apolypeptide comprising one or more unnatural amino acid. The mutationprocess can, of course, additionally alter one or more standard codon,thereby changing one or more standard amino acid in the resulting mutantprotein, as well. Homology is generally inferred from sequencesimilarity between two or more nucleic acids or proteins (or sequencesthereof). The precise percentage of similarity between sequences that isuseful in establishing homology varies with the nucleic acid and proteinat issue, but as little as 25% sequence similarity is routinely used toestablish homology. Higher levels of sequence similarity, e.g., 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more, can also be used toestablish homology. Methods for determining sequence similaritypercentages (e.g., BLASTP and BLASTN using default parameters) aredescribed herein and are generally available.

Orthogonal: As used herein, the term “orthogonal” refers to a molecule(e.g., an orthogonal tRNA (O-tRNA) and/or an orthogonal aminoacyl tRNAsynthetase (O-RS)) that functions with endogenous components of a cellwith reduced efficiency as compared to a corresponding molecule that isendogenous to the cell or translation system, or that fails to functionwith endogenous components of the cell. In the context of tRNA's andaminoacyl-tRNA synthetases, orthogonal refers to an inability or reducedefficiency, e.g., less than 20% efficient, less than 10% efficient, lessthan 5% efficient, or less than 1% efficient, of an orthogonal tRNA tofunction with an endogenous tRNA synthetase compared to an endogenoustRNA to function with the endogenous tRNA synthetase, or of anorthogonal aminoacyl-tRNA synthetase to function with an endogenous tRNAcompared to an endogenous tRNA synthetase to function with theendogenous tRNA. The orthogonal molecule lacks a functional endogenouscomplementary molecule in the cell. For example, an orthogonal tRNA in acell is aminoacylated by any endogenous RS of the cell with reduced oreven zero efficiency, when compared to aminoacylation of an endogenoustRNA by the endogenous RS. In another example, an orthogonal RSaminoacylates any endogenous tRNA in a cell of interest with reduced oreven zero efficiency, as compared to aminoacylation of the endogenoustRNA by an endogenous RS. A second orthogonal molecule can be introducedinto the cell that functions with the first orthogonal molecule. Forexample, an orthogonal tRNA/RS pair includes introduced complementarycomponents that function together in the cell with an efficiency (e.g.,50% efficiency, 60% efficiency, 70% efficiency, 75% efficiency, 80%efficiency, 90% efficiency, 95% efficiency, or 99% or more efficiency)to that of a corresponding tRNA/RS endogenous pair.

Complementary: The term “complementary” refers to components of anorthogonal pair, O-tRNA and O-RS that can function together, e.g., wherethe O-RS aminoacylates the O-tRNA.

Preferentially Aminoacylates: The term “preferentially aminoacylates”refers to an efficiency, e.g., 70% efficient, 75% efficient, 85%efficient, 90% efficient, 95% efficient, or 99% or more efficient, atwhich an O-RS aminoacylates an O-tRNA with an unnatural amino acid ascompared to the O-RS aminoacylating a naturally occurring tRNA or astarting material used to generate the O-tRNA. The unnatural amino acidis incorporated into a growing polypeptide chain with high fidelity,e.g., at greater than 75% efficiency for a given selector codon, atgreater than about 80% efficiency for a given selector codon, at greaterthan about 90% efficiency for a given selector codon, at greater thanabout 95% efficiency for a given selector codon, or at greater thanabout 99% or more efficiency for a given selector codon.

Selector Codon: The term “selector codon” refers to codons recognized bythe O-tRNA in the translation process and not recognized by anendogenous tRNA. The O-tRNA anticodon loop recognizes the selector codonon the mRNA and incorporates its amino acid, e.g., an unnatural aminoacid, at this site in the polypeptide. Selector codons can include,e.g., nonsense codons, such as, stop codons, e.g., amber, ochre, andopal codons; four or more base codons; rare codons; codons derived fromnatural or unnatural base pairs and/or the like.

Suppressor tRNA: A suppressor tRNA is a tRNA that alters the reading ofa messenger RNA (mRNA) in a given translation system, e.g., by providinga mechanism for incorporating an amino acid into a polypeptide chain inresponse to a selector codon. For example, a suppressor tRNA can readthrough, e.g., a stop codon, a four base codon, a rare codon, and/or thelike.

Recyclable tRNA: The term “recyclable tRNA” refers to a tRNA that isaminoacylated and can be repeatedly reaminoacylated with an amino acid(e.g., an unnatural amino acid) for the incorporation of the amino acid(e.g., the unnatural amino acid) into one or more polypeptide chainsduring translation.

Translation System: The term “translation system” refers to thecollective set of components that incorporate a naturally occurringamino acid into a growing polypeptide chain (protein). Components of atranslation system can include, e.g., ribosomes, tRNA's, synthetases,mRNA, amino acids, and the like. The components of the invention (e.g.,ORS, OtRNA's, unnatural amino acids, etc.) can be added to an in vitroor in vivo translation system, e.g., a vertebrate cell, e.g., a yeastcell, a mammalian cell, a plant cell, an algae cell, a fungus cell, aninsect cell, and/or the like.

Unnatural Amino Acid: As used herein, the term “unnatural amino acid”refers to any amino acid, modified amino acid, and/or amino acidanalogue that is not one of the 20 common naturally occurring aminoacids, seleno cysteine or pyrrolysine.

Derived from: As used herein, the term “derived from” refers to acomponent that is isolated from or made using information from aspecified molecule or organism.

Inactive RS: As used herein, the term “inactive RS” refers to asynthetase that has been mutated so that it no longer can aminoacylateits natural cognate tRNA with an amino acid.

Positive Selection or Screening Marker: As used herein, the term“positive selection or screening marker” refers to a marker that whenpresent, e.g., expressed, activated or the like, results inidentification of a cell with the positive selection marker from thosewithout the positive selection marker.

Negative Selection or Screening Marker: As used herein, the term“negative selection or screening marker” refers to a marker that whenpresent, e.g., expressed, activated or the like, allows identificationof a cell that does not possess the desired property (e.g., as comparedto a cell that does possess the desired property).

Reporter: As used herein, the term “reporter” refers to a component thatcan be used to select target components of a system of interest. Forexample, a reporter can include a fluorescent screening marker (e.g.,green fluorescent protein), a luminescent marker (e.g., a fireflyluciferase protein), an affinity based screening marker, or selectablemarker genes such as his3, ura3, leu2, lys2, lacZ, β-gal/lacZ(β-galactosidase), Adh (alcohol dehydrogenase), or the like.

Vertebrate: As used herein, the term “vertebrate” refers to organismsbelonging to the phylogenetic domain Eucarya such as animals e.g.,mammals, reptiles, birds, etc.

Non-eukaryote: As used herein, the term “non-eukaryote” refers tonon-vertebrate organisms. For example, a non-vertebrate organism canbelong to the Eubacteria (e.g., Escherichia coli, Thermus thermophilus,Bacillus stearothermophilus, etc.) phylogenetic domain, or the Archaea(e.g., Methanococcus jannaschii, Methanobacterium thermoautotrophicum,Halobacterium such as Haloferax volcanii and Halobacterium speciesNRC-1, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcushorikoshii, Aeuropyrum pernix etc.) phylogenetic domain.

Antibody: The term “antibody,” as used herein, includes, but is notlimited to a polypeptide substantially encoded by an immunoglobulin geneor immunoglobulin genes, or fragments thereof, which specifically bindand recognize an analyte (antigen). Examples include polyclonal,monoclonal, chimeric, and single chain antibodies, and the like.Fragments of immunoglobulins, including Fab fragments and fragmentsproduced by an expression library, including phage display, are alsoincluded in the term “antibody” as used herein. See, e.g., Paul,Fundamental Immunology, 4th Ed., 1999, Raven Press, New York, forantibody structure and terminology.

Conservative Variant: The term “conservative variant” refers to atranslation component, e.g., a conservative variant O-tRNA or aconservative variant O-RS, that functionally performs like the componentfrom which the conservative variant is based, e.g., an O-tRNA or O-RS,but has variations in the sequence. For example, an O-RS willaminoacylate a complementary O-tRNA or a conservative variant O-tRNAwith an unnatural amino acid, although the O-tRNA and the conservativevariant O-tRNA do not have the same sequence. The conservative variantcan have, e.g., one variation, two variations, three variations, fourvariations, or five or more variations in sequence, as long as theconservative variant is complementary to the corresponding O-tRNA orO-RS.

Selection or Screening Agent: As used herein, the term “selection orscreening agent” refers to an agent that, when present, allows for aselection/screening of certain components from a population. Forexample, a selection or screening agent includes, but is not limited to,e.g., a nutrient, an antibiotic, a wavelength of light, an antibody, anexpressed polynucleotide (e.g., a transcriptional modulator protein), orthe like. The selection agent can be varied, e.g., by concentration,intensity, etc.

Detectable Substance: The term “detectable substance,” as used herein,refers to an agent that, when activated, altered, expressed or the like,allows for the selection/screening of certain components from apopulation. For example, the detectable substance can be a chemicalagent, e.g., 5-fluroorotic acid (5-FOA), which under certain conditions,e.g., expression of a URA3 reporter, becomes detectable, e.g., a toxicproduct that kills cells that express the URA3 reporter.

The ability to genetically modify the structures of proteins directly invertebrate cells, beyond the chemical constraints imposed by the geneticcode, would provides a powerful molecular tool to both probe andmanipulate cellular processes. The invention provides translationalcomponents that expand the number of genetically encoded amino acids invertebrate cells. These include tRNA's (e.g., orthogonal tRNA's(O-tRNA's)), aminoacyl-tRNA synthetases (e.g., orthogonal synthetase(O-RS)), pairs of O-tRNA/O-RSs, and unnatural amino acids.

Typically, O-tRNA's of the invention are expressed and processedefficiently, and function in translation in a vertebrate cell, but arenot significantly aminoacylated by the host's aminoacyl-tRNAsynthetases. In response to a selector codon, an O-tRNA of the inventiondelivers an unnatural amino acid, which does not encode any of thecommon twenty amino acids, to a growing polypeptide chain during mRNAtranslation.

An O-RS of the invention preferentially aminoacylates an O-tRNA of theinvention with an unnatural amino acid in a vertebrate cell, but doesnot aminoacylate any of the cytoplasmic host's tRNA's. Moreover, thespecificity of an aminoacyl-tRNA synthetase of the invention providesacceptance of an unnatural amino acid while excluding any endogenousamino acids. Polypeptides that include amino acid sequences of exampleO-RSs, or portions thereof are also a feature of the invention. Inaddition, polynucleotides that encode translational components,O-tRNA's, O-RSs and portions thereof, are features of the invention.

The invention also provides methods of producing the desiredtranslational components, e.g., O-RS, and or an orthogonal pair(orthogonal tRNA and orthogonal aminoacyl-tRNA synthetase), thatutilizes an unnatural amino acid for use in a vertebrate cell (andtranslational components produced by such methods). For example, atyrosyl-tRNA synthetase/tRNA_(CUA) pair from E. coli is an O-tRNA/O-RSpair of the invention. In addition, the invention also features methodsof selecting/screening translational components in one vertebrate cell,and once selected/screened, using those components in a differentvertebrate cell (a vertebrate cell that was not used forselection/screening). For example, the selection/screening methods toproduce the translation components for vertebrate cells can be done inyeast, e.g., Saccharomyces cerevisiae, and then those selectedcomponents can be used in another vertebrate cell, e.g., another yeastcell, a mammalian cell, an insect cell, a plant cell, a fungus cell,etc.

The invention further provides methods for producing a protein in avertebrate cell, where the protein comprises an unnatural amino acid.The protein is produced using the translation components of theinvention. The invention also provides proteins (and proteins producedby the methods of the invention), which include unnatural amino acids.The protein or polypeptide of interest can also include apost-translational modification, e.g., that is added through a [3+2]cycloaddition, or a nucleophilic-electrophilic reaction, that is notmade by a prokaryotic cell, etc. In certain embodiments, methods ofproducing a transcriptional modulator protein with an unnatural aminoacid (and proteins produced by such methods) are also included in theinvention. Compositions, which include proteins that include anunnatural amino acid is also a feature of the invention.

Kits for producing a protein or polypeptide with an unnatural amino acidare also a feature of the invention.

Orthogonal Aminoacyl-TRNA Synthetases (O-RS)

In order to specifically incorporate an unnatural amino acid in to aprotein or polypeptide of interest, in a vertebrate cell, the substratespecificity of the synthetase is altered so that only the desiredunnatural amino acid, but not any of the common 20 amino acids arecharged to the tRNA. If the orthogonal synthetase is promiscuous, itwill result in mutant proteins with a mixture of natural and unnaturalamino acids at the target position. The invention provides compositionsof, and methods of, producing orthogonal aminoacyl-tRNA synthetases thathave modified substrate specificity for a specific unnatural amino acid.

A vertebrate cell that includes an orthogonal aminoacyl-tRNA synthetase(O-RS) is a feature of the invention. The O-RS preferentiallyaminoacylates an orthogonal tRNA (O-tRNA) with an unnatural amino acidin the vertebrate cell. In certain embodiments, the O-RS utilizes morethan one unnatural amino acid, e.g., two or more, three or more, etc.Thus, an O-RS of the invention can have the capability to preferentiallyaminoacylate an O-tRNA with different unnatural amino acids. This allowsan additional level of control by selecting which unnatural amino acidor combination of unnatural amino acids are put with the cell and/or byselecting the different amounts of unnatural amino acids that are putwith the cell for their incorporation.

An O-RS of the invention optionally has one or more improved or enhancedenzymatic properties for the unnatural amino acid as compared to anatural amino acid. These properties include, e.g., higher Km, lower Km,higher kcat, lower kcat, lower kcat/km, higher kcat/km, etc., for theunnatural amino acid, as compared to a naturally occurring amino acid,e.g., one of the 20 known common amino acids.

Optionally, the O-RS can be provided to the vertebrate cell by apolypeptide that includes an O-RS and/or by a polynucleotide thatencodes an O-RS or a portion thereof. For example, an O-RS, or a portionthereof, is encoded by a polynucleotide sequence as set forth in any oneof SEQ ID NO.: 3-35, or a complementary polynucleotide sequence thereof.In another example, an O-RS comprises an amino acid sequence as setforth in any one of SEQ ID NO.: 36-63, and/or 86, or a conservativevariation thereof. See, e.g., Tables 5, 6 and 8, and Example 6 hereinfor sequences of exemplary O-RS molecules.

An O-RS can also comprise an amino acid sequence that is, e.g., at least90%, at least 95%, at least 98%, at least 99%, or even at least 99.5%identical to that of a naturally occurring tyrosyl aminoacyl-tRNAsynthetase (TyrRS) (e.g., as set forth in SEQ ID NO.:2) and comprisestwo or more amino acids of group A-E. Group A includes valine,isoleucine, leucine, glycine, serine, alanine, or threonine at aposition corresponding to Tyr37 of E. coli TyrRS; group B includesaspartate at a position corresponding to Asn126 of E. coli TyrRS; groupC includes threonine, serine, arginine, aspuragine or glycine at aposition corresponding to Asp182 of E. coli TyrRS; group D includesmethionine, alanine, valine, or tyrosine at a position corresponding toPhe183 of E. coli TyrRS; and, group E includes serine, methionine,valine, cysteine, threonine, or alanine at a position corresponding toLeu186 of E. coli TyrRS.

Besides the O-RS, a vertebrate cell of the invention can includeadditional components, e.g., an unnatural amino acid(s). The vertebratecell also includes an orthogonal tRNA (O-tRNA) (e.g., derived from anon-vertebrate organism, such as Escherichia coli, Bacillusstearothermophilus, and/or the like), where the O-tRNA recognizes aselector codon and is preferentially aminoacylated with the unnaturalamino acid by the O-RS. A nucleic acid that comprises a polynucleotidethat encodes a polypeptide of interest, wherein the polynucleotidecomprises a selector codon that is recognized by the O-tRNA, or acombination of one or more of these, can also be present in the cell.

In one aspect, the O-tRNA mediates the incorporation of the unnaturalamino acid into a protein with, e.g., at least 45%, at least 50%, atleast 60%, at least 75%, at least 80%, at least 90%, at least 95%, or99% or the efficiency of as a tRNA that comprises or is processed from apolynucleotide sequence as set forth in SEQ ID NO.: 65. In anotheraspect, the O-tRNA comprises SEQ ID NO.:65, and the O-RS comprises apolypeptide sequence set forth in any one of SEQ ID NO.: 36-63, and/or86, and/or a conservative variation thereof. See also, e.g., Table 5 andExample 6, herein, for sequences of exemplary O-RS and O-tRNA molecules.

In one example, a vertebrate cell comprises an orthogonal aminoacyl-tRNAsynthetase (O-RS), an orthogonal tRNA (O-tRNA), an unnatural amino acid,and a nucleic acid that comprises a polynucleotide that encodes apolypeptide of interest, which polynucleotide comprises a selector codonthat is recognized by the O-tRNA. The O-RS preferentially aminoacylatesthe orthogonal tRNA (O-tRNA) with the unnatural amino acid in thevertebrate cell, and the cell produces the polypeptide of interest inthe absence of the unnatural amino acid with a yield that is, e.g., lessthan 30%, less than 20%, less than 15%, less than 10%, less than 5%,less than 2.5%, etc., of the yield of the polypeptide in the presence ofthe unnatural amino acid.

Methods for producing an O-RS, which are a feature of the invention,optionally include generating a pool of mutant synthetases from theframework of a wild-type synthetase, and then selecting for mutated RSsbased on their specificity for an unnatural amino acid relative to thecommon twenty amino acids. To isolate such a synthetase, the selectionmethods of the are: (i) sensitive, as the activity of desiredsynthetases from the initial rounds can be low and the population small;(ii) “tunable”, since it is desirable to vary the selection stringencyat different selection rounds; and, (iii) general, so that the methodscan be used for different unnatural amino acids.

Methods of producing an orthogonal aminoacyl-tRNA synthetase (O-RS) thatpreferentially aminoacylates an orthogonal tRNA with an unnatural aminoacid in a vertebrate cell typically include applying a combination of apositive selection followed by a negative selection. In the positiveselection, suppression of the selector codon introduced at nonessentialposition(s) of a positive marker allows the vertebrate cells to surviveunder positive selection pressure. In the presence of unnatural aminoacids, survivors thus encode active synthetases charging the orthogonalsuppressor tRNA with an unnatural amino acid. In the negative selection,suppression of a selector codon introduced at nonessential position(s)of a negative marker removes synthetases with natural amino acidspecificities. Survivors of the negative and positive selection encodesynthetases that aminoacylate (charge) the orthogonal suppressor tRNAwith unnatural amino acids only (or at least preferentially).

For example, the method includes: (a) subjecting to positive selection,in the presence of an unnatural amino acid, a population of vertebratecells of a first species, where the vertebrate cells each comprise: i) amember of a library of aminoacyl-tRNA synthetases (RSs), ii) anorthogonal tRNA (O-tRNA), iii) a polynucleotide that encodes a positiveselection marker, and iv) a polynucleotide that encodes a negativeselection marker, wherein cells that survive the positive selectioncomprise an active RS that aminoacylates the orthogonal tRNA (O-tRNA) inthe presence of an unnatural amino acid; and, (b) subjecting the cellsthat survive the positive selection to negative selection in the absenceof the unnatural amino acid to eliminate active RSs that aminoacylatethe O-tRNA with a natural amino acid, thereby providing the O-RS thatpreferentially aminoacylates the O-tRNA with the unnatural amino acid.

The positive selection marker can be any of a variety of molecules. Inone embodiment, the positive selection marker is a product that providesa nutritional supplement for growth and the selection is performed on amedium that lacks the nutritional supplement. Examples ofpolynucleotides that encode positive selection markers include, but arenot limited to, e.g., a reporter gene based on complementing the aminoacid auxotrophy of a cell, a his3 gene (e.g., where the his3 geneencodes an imidazole glycerol phosphate dehydratase, detected byproviding 3-aminotriazole (3-AT)), ura3 gene, leu2 gene, lys2 gene, lacZgene, adh gene, etc. See, e.g., G. M. Kishore, & D. M. Shah, (1988),Amino acid biosynthesis inhibitors as herbicides, Annual Review ofBiochemistry 57:627-663. In one embodiment, lacZ production is detectedby ortho-nitrophenyl-β-D-galactopyranoside (ONPG) hydrolysis. See, e.g.,I. G. Serebriiskii, & E. A. Golemis, (2000), Uses of lacZ to study genefunction: evaluation of beta-galactosidase assays employed in the yeasttwo-hybrid system, Analytical Biochemistry 285:1-15. Additional positiveselection markers include, e.g., luciferase, green fluorescent protein(GFP), YFP, EGFP, RFP, the product of an antibiotic resistant gene(e.g., chloramphenicol acetyltransferase (CAT)), a transcriptionalmodulator protein (e.g., GAL4), etc. Optionally, a polynucleotide thatencodes a positive selection marker comprises a selector codon.

A polynucleotide that encodes the positive selection marker can beoperably linked to a response element. An additional polynucleotide thatencodes a transcriptional modulator protein that modulates transcriptionfrom the response element, and comprises at least one selector codon,can also be present. The incorporation of the unnatural amino acid intothe transcriptional modulator protein by the O-tRNA aminoacylated withthe unnatural amino acid results in transcription of the polynucleotide(e.g., reporter gene) encoding the positive selection marker.Optionally, the selector codon is located in or substantially near aportion of the polynucleotide that encodes a DNA binding domain of thetranscriptional modulator protein.

A polynucleotide that encodes the negative selection marker can also beoperably linked to a response element from which transcription ismediated by the transcriptional modulator protein. See, e.g., A. J.DeMaggio, et al., (2000), The yeast split-hybrid system, Method Enzymol.328:128-137; H. M. Shih, et al., (1996), A positive genetic selectionfor disrupting protein-protein interactions: identiflcation of CREBmutations that prevent association with the coactivator CBP, Proc. Natl.Acad. Sci. U.S.A. 93:13896-13901; M. Vidal, et al., (1996), Geneticcharacterization of a mammalian protein-protein interaction domain byusing a yeast reverse two-hybrid system. [comment], Proc. Natl. Acad.Sci. U.S.A. 93:10321-10326; and, M. Vidal, et al., (1996), Reversetwo-hybrid and one-hybrid systems to detect dissociation ofprotein-protein and DNA-protein interactions. [comment], Proc. Natl.Acad. Sci. U.S.A. 93:10315-10320. The incorporation of a natural aminoacid into the transcriptional modulator protein by the O-tRNAaminoacylated with a natural amino acid results in transcription of thenegative selection marker. Optionally, the negative selection markercomprises a selector codon. In one embodiment, the positive selectionmarker and/or negative selection marker of the invention can comprise atleast two selector codons, which each or both can comprise at least twodifferent selector codons or at least two of the same selector codons.

The transcriptional modulator protein is a molecule that binds (directlyor indirectly) to a nucleic acid sequence (e.g., a response element) andmodulates transcription of a sequence that is operably linked to theresponse element. A transcriptional modulator protein can be atranscriptional activator protein (e.g., GAL4, nuclear hormonereceptors, AP1, CREB, LEF/tcf family members, SMADs, VP16, SP1, etc.), atranscriptional repressor protein (e.g., nuclear hormone receptors,Groucho/tle family, Engrailed family, etc), or a protein that can haveboth activities depending on the environment (e.g., LEF/tcf, homoboxproteins, etc.). A response element is typically a nucleic acid sequencethat is recognized by the transcriptional modulator protein or anadditional agent that acts in concert with the transcriptional modulatorprotein.

Another example of a transcriptional modulator protein is thetranscriptional activator protein, GAL4. See, e.g., A. Laughon, et al.,(1984), Identification of two proteins encoded by the Saccharomycescerevisiae GAL4 gene, Molecular & Cellular Biology 4:268-275; A.Laughon, & R. F. Gesteland, (1984), Primary structure of theSaccharomyces cerevisiae GAL4 gene, Molecular & Cellular Biology4:260-267; L. Keegan, et al., (1986), Separation of DNA binding from thetranscription-activating function ofa vertebrate regulatory protein,Science 231:699-704; and, M. Ptashne, (1988), How vertebratetranscriptional activators work, Nature 335:683-689. The N-terminal 147amino acids of this 881 amino acid protein form a DNA binding domain(DBD) that binds DNA sequence specifically. See, e.g., M. Carey, et al.,(1989), An amino-terminal fragment of GAL4 binds DNA as a dimer, J. Mol.Biol. 209:423-432; and, E. Giniger, et al., (1985), Specific DNA bindingof GAL4, a positive regulatory protein of yeast, Cell 40:767-774. TheDBD is linked, by an intervening protein sequence, to a C-terminal 113amino acid activation domain (AD) that can activate transcription whenbound to DNA. See, e.g., J. Ma, & M. Ptashne, (1987), Deletion analysisof GAL4 defines two transcriptional activating segments, Cell48:847-853: and, J. Ma, & M. Ptashne, (1987), The carboxy-terminal 30amino acids of GAL4 are recognized by GAL80, Cell 50:137-142. By placingamber codons towards, e.g., the N-terminal DBD of a single polypeptidethat contains both the N-terminal DBD of GAL4 and its C-terminal AD,amber suppression by the O-tRNA/O-RS pair can be linked totranscriptional activation by GAL4. GAL4 activated reporter genes can beused to perform both positive and negative selections with the gene.

The medium used for negative selection can comprise a selecting orscreening agent that is converted to a detectable substance by thenegative selection marker. In one aspect of the invention, thedetectable substance is a toxic substance. A polynucleotide that encodesa negative selection marker can be, e.g., an ura3 gene. For example, theURA3 reporter can be placed under control of a promoter that containsGAL4 DNA binding sites. When the negative selection marker is produced,e.g., by translation of a polynucleotide encoding the GAL4 with selectorcodons, GAL4 activates transcription of URA3. The negative selection isaccomplished on a medium that comprises 5-fluoroorotic acid (5-FOA),which is converted into a detectable substance (e.g., a toxic substancewhich kills the cell) by the gene product of the ura3 gene. See, e.g.,J. D. Boeke, et al., (1984), A positive selection for mutants lackingorotidine-5′-phosphate decarboxylase activity in yeast: 5-fluorooroticacid resistance, Molecular & General Genetics 197:345-346); M. Vidal, etal., (1996), Genetic characterization of a mammalian protein-proteininteraction domain by using a yeast reverse two-hybrid system.[comment], Proc. Natl. Acad. Sci. U.S.A. 93:10321-10326; and, M. Vidal,et al., (1996), Reverse two-hybrid and one-hybrid systems to detectdissociation of protein-protein and DNA-protein interactions. [comment],Proc. Natl. Acad. Sci. U.S.A. 93:10315-10320.

As with the positive selection marker, the negative selection marker canalso be any of a variety of molecules. In one embodiment, the positiveselection marker and/or the negative selection marker is a polypeptidethat fluoresces or catalyzes a luminescent reaction in the presence of asuitable reactant. For example, negative selection markers include, butare not limited to, e.g., luciferase, green fluorescent protein (GFP),YFP, EGFP, RFP, the product of an antibiotic resistant gene (e.g.,chloramphenicol acetyltransferase (CAT)), the product of a lacZ gene,transcriptional modulator protein, etc. In one aspect of the invention,the positive selection marker and/or the negative selection marker isdetected by fluorescence-activated cell sorting (FACS) or byluminescence. In another example, the positive selection marker and/ornegative selection marker comprise an affinity based screening marker.The same polynucleotide can encode both the positive selection markerand the negative selection marker.

Additional levels of selection/screening stringency can also be used inthe methods of the invention. The selection or screening stringency canbe varied on one or both steps of the method to produce an O-RS. Thiscould include, e.g., varying the amount of response elements in apolynucleotide that encodes the positive and/or negative selectionmarker, adding a varying amount of an inactive synthetase to one or bothof the steps, varying the amount of selection/screening agent that isused, etc. Additional rounds of positive and/or negative selections canalso be performed.

Selecting or screening can also comprise one or more positive ornegative selection or screening that includes, e.g., a change in aminoacid permeability, a change in translation efficiency, a change intranslational fidelity, etc. Typically, the one or more change is basedupon a mutation in one or more polynucleotides that comprise or encodecomponents of an orthogonal tRNA-tRNA synthetase pair that are used toproduce protein.

Model enrichment studies can also be used to rapidly select an activesynthetase from an excess of inactive synthetases. Positive and/ornegative model selection studies can be done. For example, vertebratecells that comprise potential active aminoacyl-tRNA synthetases aremixed with a varying fold excess of inactive aminoacyl-tRNA synthetases.A ratio comparison is made between cells grown in a nonselective mediaand assayed by, e.g., X-GAL overlay, and those grown and able to survivein a selective media (e.g., in the absence of histidine and/or uracil)and assayed by, e.g., an X-GAL assay. For a negative model selection,potential active aminoacyl-tRNA synthetases are mixed with a varyingfold excess of inactive aminoacyl-tRNA synthetases and selection isperformed with a negative selection substance, e.g., 5-FOA.

Typically, the library of RSs (e.g., a library of mutant RSs) comprisesRSs derived from at least one aminoacyl-tRNA synthetase (RS), e.g., froma non-vertebrate organism. In one embodiment, the library of RSs isderived from an inactive RS, e.g., where the inactive RS is generated bymutating an active RS, e.g., at the active site in the synthetase, atthe editing mechanism site in the synthetase, at different sites bycombining different domains of synthetases, or the like. For example,residues in the active site of the RS are mutated to, e.g., alanineresidues. The polynucleotide that encodes the alanine mutated RS is usedas a template to mutagenize the alanine residues to all 20 amino acids.The library of mutant RSs is selected/screened to produce the O-RS. Inanother embodiment, the inactive RS comprises an amino acid bindingpocket and one or more amino acids that comprise the binding pocket aresubstituted with one or more different amino acids. In one example, thesubstituted amino acids are substituted with alanines. Optionally, thepolynucleotide that encodes the alanine mutated RS is used as a templateto mutagenize the alanine residues to all 20 amino acids andscreened/selected.

The method of producing an O-RS can further include producing thelibrary of RSs by using various mutagenesis techniques known in the art.For example, the mutant RSs can be generated by site-specific mutations,random point mutations, homologous recombination, DNA shuffling or otherrecursive mutagenesis methods, chimeric construction or any combinationthereof. For example, a library of mutant RSs can be produced from twoor more other, e.g., smaller, less diverse “sub-libraries.” Once thesynthetases are subjected to the positive and negativeselection/screening strategy, these synthetases can then be subjected tofurther mutagenesis. For example, a nucleic acid that encodes the O-RScan be isolated; a set of polynucleotides that encode mutated O-RSs(e.g., by random mutagenesis, site-specific mutagenesis, recombinationor any combination thereof) can be generated from the nucleic acid; and,these individual steps or a combination of these steps can be repeateduntil a mutated O-RS is obtained that preferentially aminoacylates theO-tRNA with the unnatural amino acid. In one aspect of the invention,the steps are performed at least two times.

Additional details for producing O-RS can be found in WO 2002/086075entitled “Methods and compositions for the production of orthogonaltRNA-aminoacyltRNA synthetase pairs.” See also, Hamano-Takaku et al,(2000) A mutant Escherichia coli Tyrosyl-tRNA Synthetase Utilizes theUnnatural Amino Acid Azatyrosine More Efficiently than yrosine, Journalof Biological Chemistry. 275(51):40324-40328; Kiga et al. (2002), Anengineered Escherichia coli tyrosyl-tRNA synthetase for site-specificincorporation of an unnatural amino acid into proteins in vertebratetranslation and its application in a wheat germ cell-free system, PNAS99(15): 9715-9723; and, Francklyn et al., (2002), Aminoacyl-tRNAsynthetases: Versatile players in the changing theater of translation;RNA 8:1363-1372.

Orthogonal tRNA's

Eukaryotic cells that include an orthogonal tRNA (O-tRNA) are providedby the invention. The orthogonal tRNA mediates incorporation of anunnatural amino acid into a protein that is encoded by a polynucleotidethat comprises a selector codon that is recognized by the O-tRNA, invivo. In certain embodiments, an O-tRNA of the invention mediates theincorporation of an unnatural amino acid into a protein with, e.g., atleast 40%, at least 45%, at least 50%, at least 60%, at least 75%, atleast 80%, or even 90% or more as efficiently as tRNA that comprises oris processed in a cell from a polynucleotide sequence as set forth inSEQ ID NO.: 65. See, Table 5, herein.

An example of an O-tRNA of the invention is SEQ ID NO.: 65. (See Example6 and Table 5, herein). SEQ ID NO.: 65 is a pre-splicing/processingtranscript that is optionally processed in the cell, e.g., using thestandard endogenous cellular splicing and processing machinery, andmodified to form an active O-tRNA. Typically, a population of suchpre-splicing transcripts forms a population of active tRNA's in thecell. The invention also includes conservative variations of the O-tRNAand its processed cellular products. For example, conservativevariations of O-tRNA include those molecules that function like theO-tRNA of SEQ ID NO.:65 and maintain the tRNA L-shaped structure inprocessed form, but do not have the same sequence (and are other thanwild type tRNA molecules). Typically, an O-tRNA of the invention is arecyclable O-tRNA, because the O-tRNA can be reaminoacylated in vivo toagain mediate the incorporation of the unnatural amino acid into aprotein that is encoded by a polynucleotide in response to a selectorcodon.

The transcription of the tRNA in eukaryotes, but not in prokaryotes, iscarried out by RNA Polymerase III, which places restrictions on theprimary sequence of the tRNA structural genes that can be transcribed invertebrate cells. In addition, in vertebrate cells, tRNA's need to beexported from the nucleus, where they are transcribed, to the cytoplasm,to function in translation. Nucleic acids that encode an O-tRNA of theinvention or a complementary polynucleotide thereof are also a featureof the invention. In one aspect of the invention, a nucleic acid thatencodes an O-tRNA of the invention includes an internal promotersequence, e.g., an A box (e.g., TRGCNNAGY) and a B box (e.g.,GGTTCGANTCC, SEQ ID NO: 95). Additional examples of A box and B boxsequences can be found in Geiduschek, (1988), Transcription By RNAPolymerase III, Ann. Rev, Biochem. 57:873-914, which is incorporated byreference herein. The O-tRNA of the invention can also bepost-transcriptionally modified. For example, post-transcriptionalmodification of tRNA genes in eukaryotes includes removal of the 5′- and3′-flanking sequences by Rnase P and a 3′-endonuclease, respectively.The addition of a 3′-CCA sequence is also a post-transcriptionalmodification of a tRNA gene in eukaryotes.

In one embodiment, an O-tRNA is obtained by subjecting to negativeselection a population of vertebrate cells of a first species, where thevertebrate cells comprise a member of a library of tRNA's. The negativeselection eliminates cells that comprise a member of the library oftRNA's that is aminoacylated by an aminoacyl-tRNA synthetase (RS) thatis endogenous to the vertebrate cells. This provides a pool of tRNA'sthat are orthogonal to the vertebrate cell of the first species.

Alternatively, or in combination with others methods described above toincorporate an unnatural amino acid into a polypeptide, atrans-translation system can be used. This system involves a moleculecalled tmRNA present in Escherichia coli. This RNA molecule isstructurally related to an alanyl tRNA and is aminoacylated by thealanyl synthetase. The difference between tmRNA and tRNA is that theanticodon loop is replaced with a special large sequence. This sequenceallows the ribosome to resume translation on sequences that have stalledusing an open reading frame encoded within the tmRNA as template. In theinvention, an orthogonal tmRNA can be generated that is preferentiallyaminoacylated with an orthogonal synthetase and loaded with an unnaturalamino acid. By transcribing a gene by the system, the ribosome stalls ata specific site; the unnatural amino acid is introduced at that site,and translation resumes using the sequence encoded within the orthogonaltmRNA.

Additional methods for producing a recombinant orthogonal tRNA's can befound, e.g., in International patent applications WO 2002/086075,entitled “Methods and compositions for the production of orthogonaltRNA-aminoacyltRNA synthetase pairs.” See also, Forster et al., (2003)Programming peptidomimetic synthetases by translating genetic codesdesigned de novo PNAS 100(11):6353-6357; and, Feng et al., (2003),Expanding tRNA recognition of a tRNA synthetase by a single amino acidchange, PNAS 100(10): 5676-5681.

Orthogonal TRNA and Orthogonal Aminoacyl-TRNA Synthetase Pairs

An orthogonal pair is composed of an O-tRNA, e.g., a suppressor tRNA, aframeshift tRNA, or the like, and an O-RS. The O-tRNA is not acylated byendogenous synthetases and is capable of mediating incorporation of anunnatural amino acid into a protein that is encoded by a polynucleotidethat comprises a selector codon that is recognized by the O-tRNA invivo. The O-RS recognizes the O-tRNA and preferentially aminoacylatesthe O-tRNA with an unnatural amino acid in a vertebrate cell. Methodsfor producing orthogonal pairs along with orthogonal pairs produced bysuch methods and compositions of orthogonal pairs for use in vertebratecells are included in the invention. The development of multipleorthogonal tRNA/synthetase pairs can allow the simultaneousincorporation of multiple unnatural amino acids using different codonsin a vertebrate cell.

An orthogonal O-tRNA/O-RS pair in a vertebrate cell can be produced byimporting a pair, e.g., a nonsense suppressor pair, from a differentorganism with inefficient cross species aminoacylation. The O-tRNA andO-RS are efficiently expressed and processed in the vertebrate cell andthe O-tRNA is efficiently exported from the nucleus to the cytoplasm.For example, one such pair is the tyrosyl-tRNA synthetase/tRNA_(CUA)pair from E. coli (see, e.g., H. M. Goodman, et al., (1968), Nature217:1019-24; and, D. G. Barker, et al., (1982), FEBS Letters150:419-23). E. coli tyrosyl-tRNA synthetase efficiently aminoacylatesits cognate E. coli tRNA_(CUA) when both are expressed in the cytoplasmof S. cerevisiae, but does not aminoacylate S. cerevisiae tRNA's. See,e.g., H. Edwards, & P. Schimmel, (1990), Molecular & Cellular Biology10:1633-41; and, H. Edwards, et al., (1991), PNAS United States ofAmerica 88:1153-6. In addition, E. coli tyrosyl tRNA_(CUA) is a poorsubstrate for S. cerevisiae aminoacyl-tRNA synthetases (see, e.g., V.Trezeguet, et al., (1991), Molecular & Cellular Biology 11:2744-51), butfunctions efficiently in protein translation in S. cerevisiae. See,e.g., H. Edwards, & P. Schimmel, (1990) Molecular & Cellular Biology10:1633-41; H. Edwards, et al., (1991), PNAS United States of America88:1153-6; and, V. Trezeguet, et al., (1991), Molecular & CellularBiology 11:2744-51. Moreover, E. coli TyrRS does not have an editingmechanism to proofread an unnatural amino acid ligated to the tRNA.

The O-tRNA and O-RS can be naturally occurring or can be derived bymutation of a naturally occurring tRNA and/or RS, which generateslibraries of tRNA's and/or libraries of RSs, from a variety of organism.See the section entitled “Sources and Hosts” herein. In variousembodiments, the O-tRNA and O-RS are derived from at least one organism.In another embodiment, the O-tRNA is derived from a naturally occurringor mutated naturally occurring tRNA from a first organism and the O-RSis derived from naturally occurring or mutated naturally occurring RSfrom a second organism. In one embodiment, the first and secondnon-vertebrate organisms are the same. Alternatively, the first andsecond non-vertebrate organisms can be different.

See sections herein entitled “Orthogonal aminoacyl-tRNA synthetases” and“O-tRNA” for methods of producing O-RSs and O-tRNA's. See also,International patent application WO 2002/086075, entitled “Methods andcompositions for the production of orthogonal tRNA-aminoacyltRNAsynthetase pairs.”

Fidelity, Efficiency, and Yield

Fidelity refers to the accuracy with which a desired molecule, e.g., anunnatural amino acid or amino acid, is incorporated into a growingpolypeptide at a desired position. The translational components of theinvention incorporate unnatural amino acids, with high fidelity, intoproteins in response to a selector codon. For example, using thecomponents of the invention, the efficiency of incorporation of adesired unnatural amino acid into a growing polypeptide chain at adesired position (e.g., in response to a selector codon) is, e.g.,greater than 75%, greater than 85%, greater than 95%, or even greaterthan 99% or more as efficient as compared to unwanted incorporation aspecific natural amino acid being incorporated into the growingpolypeptide chain the desired position.

Efficiency can also refer to the degree with which the O-RSaminoacylates the O-tRNA with the unnatural amino acid as compared to arelevant control. O-RSs of the invention can be defined by theirefficiency. In certain embodiments of the invention, an O-RS is comparedto another O-RS. For example, a O-RS of the invention aminoacylates aO-tRNA with an unnatural amino acid, e.g., at least 40%, at least 50%,at least 60%, at least 75%, at least 80%, at least 90%, at least 95%, oreven 99% or more as efficiently as an O-RS having an amino acidsequence, e.g., as set forth in SEQ ID NO.: 86 or 45) or anotherspecific RS in Table 5) aminoacylates an O-tRNA. In another embodiment,an O-RS of the invention aminoacylates the O-tRNA with the unnaturalamino acid at least 10-fold, at least 20-fold, at least 30-fold, etc.,more efficiently than the O-RS aminoacylates the O-tRNA with a naturalamino acid.

Using the translational components of the invention, the yield of thepolypeptide of interest comprising the unnatural amino acid is, e.g., atleast 5%, at least 10%, at least 20%, at least 30%, at least 40%, 50% ormore, of that obtained for the naturally occurring polypeptide ofinterest from a cell in which the polynucleotide lacks the selectorcodon. In another aspect, the cell produces the polypeptide of interestin the absence of the unnatural amino acid with a yield that is, e.g.,less than 30%, less than 20%, less than 15%, less than 10%, less than5%, less than 2.5%, etc., of the yield of the polypeptide in thepresence of the unnatural amino acid.

Source and Host Organisms

The orthogonal translational components of the invention are typicallyderived from non-vertebrate organisms for use in vertebrate cells ortranslation systems. For example, the orthogonal O-tRNA can be derivedfrom a non-vertebrate organism, e.g., a eubacterium, such as Escherichiacoli, Thermus thermophilus, Bacillus stearothermphilus, or the like, oran archaebacterium, such as Methanococcus jannaschii, Methanobacteriumthermoautotrophicum, Halobacterium such as Haloferax volcanii andHalobacterium species NRC-1, Archaeoglobus fulgidus, Pyrococcusfuriosus, Pyrococcus horikoshii, Aeuropyrum pernix, or the like, whilethe orthogonal O-RS can be derived from a non-vertebrate organism, e.g.,a eubacterium, such as Escherichia coli, Thermus thermophilus, Bacillusstearothermphilus, or the like, or an arcbaebacterium, such asMethanococcus jannaschii, Methanobacterium thermoautotrophicum,Halobacterium such as Haloferax volcanii and Halobacterium speciesNRC-1, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcushorikoshii, Aeuropyrum pernix, or the like. Alternately, vertebratesources can also be used, e.g., plants, algae, protists, fungi, yeasts,animals (e.g., mammals, insects, arthropods, etc.), or the like, e.g.,where the components are orthogonal to a cell or translation system ofinterest, or where they are modified (e.g., mutated) to be orthogonal tothe cell or translation system.

The individual components of an O-tRNA/O-RS pair can be derived from thesame organism or different organisms. In one embodiment, the O-tRNA/O-RSpair is from the same organism. For example, the O-tRNA/O-RS pair can bederived from a tyrosyl-tRNA synthetase/tRNA_(CUA) pair from E. coli.Alternatively, the O-tRNA and the O-RS of the O-tRNA/O-RS pair areoptionally from different organisms.

The orthogonal O-tRNA, O-RS or O-tRNA/O-RS pair can be selected orscreened and/or used in a vertebrate cell to produce a polypeptide withan unnatural amino acid. A vertebrate cell can be from a variety ofsources, e.g., any vertebrate animal (e.g., a mammal, an amphibian,birds, reptiles, fish, etc.), or the like. Compositions of vertebratecells with translational components of the invention are also a featureof the invention.

The invention also provides for the efficient screening in one speciesfor optional use in that species and/or a second species (optionally,without additional selection/screening). For example, the components ofthe O-tRNA/O-RS are selected or screened in one species, e.g., an easilymanipulated species (such as a yeast cell, etc.) and introduced into asecond vertebrate species, e.g., a plant (e.g., complex plant such asmonocots, or dicots), an algae, a protist, a fungus, a yeast, an animal(e.g., a mammal, an insect, an arthropod, etc.), or the like, for use inthe in vivo incorporation of an unnatural amino acid in the secondspecies.

For example, Saccharomyces cerevisiae (S. cerevisiae) can be chosen asthe vertebrate first species, as it is unicellular, has a rapidgeneration time, and relatively well-characterized genetics. See, e.g.,D. Burke, et al., (2000) Methods in Yeast Genetics. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. Moreover, since thetranslational machinery of eukaryotes is highly conserved (see, e.g.,(1996) Translational Control. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.; Y. Kwok, & J. T. Wong, (1980), Evolutionary relationshipbetween Halobacterium cutirubrum and eukaryotes determined by use ofaminoacyl-tRNA synthetases as phylogenetic probes, Canadian Journal ofBiochemistry 58:213-218; and, (2001) The Ribosome, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.), aaRSs genes for theincorporation of unnatural amino acids discovered in S. cerevisiae canbe introduced into higher vertebrate organisms and used, in partnershipwith cognate tRNA's (see, e.g., K. Sakamoto, et al., (2002)Site-specific incorporation of an unnatural amino acid into proteins inmammalian cells, Nucleic Acids Res. 30:4692-4699; and, C. Kohrer, etal., (2001), Import of amber and ochre suppressor tRNA's into mammaliancells: a general approach to site-specific insertion of amino acidanalogues into proteins, Proc. Natl. Acad. Sci. U.S.A. 98:14310-14315)to incorporate unnatural amino acids.

In one example, the method of producing O-tRNA/O-RS in a first speciesas described herein further includes introducing a nucleic acid thatencodes the O-tRNA and a nucleic acid that encodes the O-RS into avertebrate cell of a second species (e.g., a mammal, an insect, afungus, an algae, a plant and the like). In another example, a method ofproducing an orthogonal aminoacyl-tRNA synthetase (O-RS) thatpreferentially aminoacylates an orthogonal tRNA with an unnatural aminoacid in a vertebrate cell includes: (a) subjecting to positiveselection, in the presence of an unnatural amino acid, a population ofvertebrate cells of a first species (e.g., yeast and the like). Each ofthe vertebrate cells comprise: i) a member of a library ofaminoacyl-tRNA synthetases (RSs), ii) an orthogonal tRNA (O-tRNA), iii)a polynucleotide that encodes a positive selection marker, and iv) apolynucleotide that encodes a negative selection marker. The cells thatsurvive the positive selection comprise an active RS that aminoacylatesthe orthogonal tRNA (O-tRNA) in the presence of an unnatural amino acid.The cells that survive the positive selection are subjected to negativeselection in the absence of the unnatural amino acid to eliminate activeRSs that aminoacylate the O-tRNA with a natural amino acid. Thisprovides an O-RS that preferentially aminoacylates the O-tRNA with theunnatural amino acid. A nucleic acid that encodes the O-tRNA and anucleic acid that encodes the O-RS (or the components O-tRNA and/orO-RS) are introduced into a vertebrate cell of a second species e.g., amammal, an insect, a fungus, an algae, a plant and/or the like.Typically, the O-tRNA is obtained by subjecting to negative selection apopulation of vertebrate cells of a first species, where the vertebratecells comprise a member of a library of tRNA's. The negative selectioneliminates cells that comprise a member of the library of tRNA's that isaminoacylated by an aminoacyl-tRNA synthetase (RS) that is endogenous tothe vertebrate cells, which provides a pool of tRNA's that areorthogonal to the vertebrate cell of the first species and the secondspecies.

Selector Codons

Selector codons of the invention expand the genetic codon framework ofthe protein biosynthetic machinery. For example, a selector codonincludes, e.g., a unique three base codon, a nonsense codon, such as astop codon, e.g., an amber codon (UAG), an opal codon (UGA), anunnatural codon, at least a four base codon, a rare codon, or the like.A number of selector codons can be introduced into a desired gene, e.g.,one or more, two or more, more than three, etc. Once gene can includemultiple copies of a given selector codon, or can include multipledifferent selector codons, or any combination thereof.

In one embodiment, the methods involve the use of a selector codon thatis a stop codon for the incorporation of unnatural amino acids in vivoin a vertebrate cell. For example, an O-tRNA is produced that recognizesthe stop codon, e.g., UAG, and is aminoacylated by an O-RS with adesired unnatural amino acid. This O-tRNA is not recognized by thenaturally occurring host's aminoacyl-tRNA synthetases. Conventionalsite-directed mutagenesis can be used to introduce the stop codon, e.g.,TAG, at the site of interest in a polypeptide of interest. See, e.g.,Sayers, J. R., et al. (1988), 5′,3′ Exonuclease inphosphorothioate-based oligonucleottde-directed mutagenesis. NucleicAcids Res. 791-802. When the O-RS, O-tRNA and the nucleic acid thatencodes the polypeptide of interest are combined in vivo, the unnaturalamino acid is incorporated in response to the UAG codon to give apolypeptide containing the unnatural amino acid at the specifiedposition.

The incorporation of unnatural amino acids in vivo can be done withoutsignificant perturbation of the vertebrate host cell. For example,because the suppression efficiency for the UAG codon depends upon thecompetition between the O-tRNA, e.g., the amber suppressor tRNA, and avertebrate release factor (e.g., eRF) (which binds to a stop codon andinitiates release of the growing peptide from the ribosome), thesuppression efficiency can be modulated by, e.g., increasing theexpression level of O-tRNA, e.g., the suppressor tRNA.

Selector codons also comprise extended codons, e.g., four or more basecodons, such as, four, five, six or more base codons. Examples of fourbase codons include, e.g., AGGA, CUAG, UAGA, CCCU and the like. Examplesof five base codons include, e.g., AGGAC, CCCCU, CCCUC, CUAGA, CUACU,UAGGC and the like. A feature of the invention includes using extendedcodons based on frameshift suppression. Four or more base codons caninsert, e.g., one or multiple unnatural amino acids into the sameprotein. For example, in the presence of mutated O-tRNA's, e.g., aspecial frameshift suppressor tRNA's, with anticodon loops, e.g., withat least 8-10 nt anticodon loops, the four or more base codon is read assingle amino acid. In other embodiments, the anticodon loops can decode,e.g., at least a four-base codon, at least a five-base codon, or atleast a six-base codon or more. Since there are 256 possible four-basecodons, multiple unnatural amino acids can be encoded in the same cellusing a four or more base codon. See, Anderson et al., (2002) Exploringthe Limits of Codon and Anticodon Size, Chemistry and Biology,9:237-244; Magliery, (2001) Expanding the Genetic Code: Selection ofEfficient Suppressors of Four-base Codons and Identification of “Shifty”Four-base Codons with a Library Approach in Escherichia coli, J. Mol,Biol. 307: 755-769.

For example, four-base codons have been used to incorporate unnaturalamino acids into proteins using in vitro biosynthetic methods. See,e.g., Ma et al., (1993) Biochemistry, 32:7939; and Hohsaka et al.,(1999) J. Am. Chem. Soc., 121:34. CGGG and AGGU were used tosimultaneously incorporate 2-naphthylalanine and an NBD derivative oflysine into streptavidin in vitro with two chemically acylatedframeshift suppressor tRNA's. See, e.g., Hobsaka et al., (1999) J. Am.Chem. Soc., 121:12194. In an in vivo study, Moore et al. examined theability of tRNALeu derivatives with NCUA anticodons to suppress UAGNcodons (N can be U, A, G, or C), and found that the quadruplet UAGA canbe decoded by a tRNALeu with a UCUA anticodon with an efficiency of 13to 26% with little decoding in the 0 or −1 frame. See, Moore et al.,(2000) J. Mol, Biol., 298:195. In one embodiment, extended codons basedon rare codons or nonsense codons can be used in invention, which canreduce missense readthrough and frameshift suppression at other unwantedsites.

For a given system, a selector codon can also include one of the naturalthree base codons, where the endogenous system does not use (or rarelyuses) the natural base codon. For example, this includes a system thatis lacking a tRNA that recognizes the natural three-base codon, and/or asystem where the three-base codon is a rare codon.

Selector codons optionally include unnatural base pairs. These unnaturalbase pairs further expand the existing genetic alphabet. One extra basepair increases the number of triplet codons from 64 to 125. Propertiesof third base pairs include stable and selective base pairing, efficientenzymatic incorporation into DNA with high fidelity by a polymerase, andthe efficient continued primer extension after synthesis of the nascentunnatural base pair. Descriptions of unnatural base pairs which can beadapted for methods and compositions include, e.g., Hirao, et al.,(2002) An unnatural base pair for incorporating amino acid analoguesinto protein, Nature Biotechnology. 20:177-182. Other relevantpublications are listed below.

For in vivo usage, the unnatural nucleoside is membrane permeable and isphosphorylated to form the corresponding triphosphate. In addition, theincreased genetic information is stable and not destroyed by cellularenzymes. Previous efforts by Benner and others took advantage ofhydrogen bonding patterns that are different from those in canonicalWatson-Crick pairs, the most noteworthy example of which is theiso-C:iso-G pair. See, e.g., Switzer et al., (1989) J. Am. Chem. Soc.,111:8322; and Piccirilli et al., (1990) Nature, 343:33; Kool, (2000)Curr. Opin. Chem. Biol., 4:602. These bases in general mispair to somedegree with natural bases and cannot be enzymatically replicated. Kooland co-workers demonstrated that hydrophobic packing interactionsbetween bases can replace hydrogen bonding to drive the formation ofbase pair. See, Kool, (2000) Curr. Onin. Chem. Biol., 4:602; and Guckianand Kool, (1998) Angew. Chem. Int. Ed. Engl., 36, 2825. In an effort todevelop an unnatural base pair satisfying all the above requirements,Schultz, Romesberg and co-workers have systematically synthesized andstudied a series of unnatural hydrophobic bases. A PICS:PICS self-pairis found to be more stable than natural base pairs, and can beefficiently incorporated into DNA by Klenow fragment of Escherichia coliDNA polymerase I (KF). See, e.g., McMinn et al., (1999) J. Am. Chem.Soc., 121:11586; and Ogawa et al., (2000) J. Am. Chem. Soc., 122:3274. A3MN:3MN self-pair can be synthesized by KF with efficiency andselectivity sufficient for biological function. See, e.g., Ogawa et al.,(2000) J. Am. Chem. Soc., 122:8803. However, both bases act as a chainterminator for further replication. A mutant DNA polymerase has beenrecently evolved that can be used to replicate the PICS self pair. Inaddition, a 7AI self pair can be replicated. See, e.g., Tae et al.,(2001) J. Am. Chem. Soc., 123:7439. A novel metallobase pair, Dipic:Py,has also been developed, which forms a stable pair upon binding Cu(II).See, Meggers et al., (2000) J. Am. Chem. Soc., 122:10714. Becauseextended codons and unnatural codons are intrinsically orthogonal tonatural codons, the methods of the invention can take advantage of thisproperty to generate orthogonal tRNA's for them.

A translational bypassing system can also be used to incorporate anunnatural amino acid in a desired polypeptide. In a translationalbypassing system, a large sequence is inserted into a gene but is nottranslated into protein. The sequence contains a structure that servesas a cue to induce the ribosome to hop over the sequence and resumetranslation downstream of the insertion.

Unnatural Amino Acids

As used herein, an unnatural amino acid refers to any amino acid,modified amino acid, or amino acid analogue other than selenocysteineand/or pyrrolysine and the following twenty genetically encodedalpha-amino acids: alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine. The generic structure of an alpha-aminoacid is illustrated by Formula I:

An unnatural amino acid is typically any structure having Formula Iwherein the R group is any substituent other than one used in the twentynatural amino acids. See, e.g., Biochemistry by L. Stryer, 3^(rd) ed.1988, Freeman and Company, New York, for structures of the twentynatural amino acids. Note that, the unnatural amino acids of theinvention can be naturally occurring compounds other than the twentyalpha-amino acids above.

Because the unnatural amino acids of the invention typically differ fromthe natural amino acids in side chain, the unnatural amino acids formamide bonds with other amino acids, e.g., natural or unnatural, in thesame manner in which they are formed in naturally occurring proteins.However, the unnatural amino acids have side chain groups thatdistinguish them from the natural amino acids. For example, R in FormulaI optionally comprises an alkyl-, aryl-, acyl-, keto-, azido-,hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynyl, ether,thiol, seleno-, sulfonyl-, borate, boronate, phospho, phosphono,phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid,hydroxylamine, amine, and the like, or any combination thereof. Otherunnatural amino acids of interest include, but are not limited to, aminoacids comprising a photoactivatable cross-linker, spin-labeled aminoacids, fluorescent amino acids, metal binding amino acids,metal-containing amino acids, radioactive amino acids, amino acids withnovel functional groups, amino acids that covalently or noncovalentlyinteract with other molecules, photocaged and/or photoisomerizable aminoacids, biotin or biotin-analogue containing amino acids, keto containingamino acids, amino acids comprising polyethylene glycol or polyether,heavy atom substituted amino acids, chemically cleavable orphotocleavable amino acids, amino acids with an elongated side chain ascompared to natural amino acids (e.g., polyethers or long chainhydrocarbons, e.g., greater than about 5, greater than about 10 carbons,etc.), carbon-linked sugar-containing amino acids, redox-active aminoacids, amino thioacid containing amino acids, and amino acids containingone or more toxic moiety. In some embodiments, the unnatural amino acidshave a photoactivatable cross-linker that is used, e.g., to link aprotein to a solid support. In one embodiment, the unnatural amino acidshave a saccharide moiety attached to the amino acid side chain (e.g.,glycosylated amino acids) and/or other carbohydrate modification.

In addition to unnatural amino acids that contain novel side chains,unnatural amino acids also optionally comprise modified backbonestructures, e.g., as illustrated by the structures of Formula II andIII:

wherein Z typically comprises OH, NH₂, SH, NH—R′, or S—R′; X and Y,which can be the same or different, typically comprise S or O, and R andR′, which are optionally the same or different, are typically selectedfrom the same list of constituents for the R group described above forthe unnatural amino acids having Formula I as well as hydrogen. Forexample, unnatural amino acids of the invention optionally comprisesubstitutions in the amino or carboxyl group as illustrated by FormulasII and III. Unnatural amino acids of this type include, but are notlimited to, α-hydroxy acids, α-thioacids α-aminothiocarboxylates, e.g.,with side chains corresponding to the common twenty natural amino acidsor unnatural side chains. In addition, substitutions at the α-carbonoptionally include L, D, or α-α-disubstituted amino acids such asD-glutamate, D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and thelike. Other structural alternatives include cyclic amino acids, such asproline analogues as well as 3, 4, 6, 7, 8, and 9 membered ring prolineanalogues, β and γ amino acids such as substituted β-alanine and γ-aminobutyric acid. For example, many unnatural amino acids are based onnatural amino acids, such as tyrosine, glutamine, phenylalanine, and thelike. Tyrosine analogs include para-substituted tyrosines,ortho-substituted tyrosines, and meta substituted tyrosines, where thesubstituted tyrosine comprises, e.g., a keto group (e.g., an acetylgroup), a benzoyl group, an amino group, a hydrazine, an hydroxyamine, athiol group, a carboxy group, an isopropyl group, a methyl group, aC₆-C₂₀ straight chain or branched hydrocarbon, a saturated orunsaturated hydrocarbon, an O-methyl group, a polyether group, a nitrogroup, an alkynyl group or the like. In addition, multiply substitutedaryl rings are also contemplated. Glutamine analogs of the inventioninclude, but are not limited to, α-hydroxy derivatives, γ-substitutedderivatives, cyclic derivatives, and amide substituted glutaminederivatives. Example phenylalanine analogs include, but are not limitedto, para-substituted phenylalanines, ortho-substituted phenyalanines,and meta-substituted phenylalanines, where the substituent comprises,e.g., a hydroxy group, a methoxy group, a methyl group, an allyl group,an aldehyde, an azido, an iodo, a bromo, a keto group (e.g., an acetylgroup), a benzoyl, an alkynyl group, or the like. Specific examples ofunnatural amino acids include, but are not limited to, ap-acetyl-L-phenylalanine, a p-propargyloxyphenylalanine,O-methyl-L,-tyrosine, an L-3-(2-naphthyl)alanine, a3-methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine,a tri-O-acetyl-GlcNAc-serine, an L-Dopa, a fluorinated phenylalanine, anisopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine,a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, ap-bromophenylalanine, a p-amino-L-phenylalanine, and anisopropyl-L-phenylalanine, and the like. Additional structures of avariety of unnatural amino acids are provided in, for example, FIGS. 16,17, 18, 19, 26, and 29 of WO 2002/085923 entitled “In vivo incorporationof unnatural amino acids.” See also, FIG. 1 structures 2-5 of Kiick etal., (2002) Incorporation of azides into recombinant proteins forchemoselective modification by the Staudinger ligtation, PNAS 99:19-24,for additional methionine analogs.

In one embodiment, compositions that include an unnatural amino acid(such as p-(propargyloxy)-phenyalanine) are provided. Variouscompositions comprising p-(propargyloxy)-phenyalanine and, e.g.,proteins and/or cells, are also provided. In one aspect, a compositionthat includes the p-(propargyloxy)-phenyalanine unnatural amino acidfurther includes an orthogonal tRNA. The unnatural amino acid can bebonded (e.g., covalently) to the orthogonal tRNA, e.g., covalentlybonded to the orthogonal tRNA though an amino-acyl bond, covalentlybonded to a 3′OH or a 2′OH of a terminal ribose sugar of the orthogonaltRNA, etc.

The chemical moieties via unnatural amino acids that can be incorporatedinto proteins offer a variety of advantages and manipulations of theprotein. For example, the unique reactivity of a keto functional groupallows selective modification of proteins with any of a number ofhydrazine- or hydroxylamine-containing reagents in vitro and in vivo. Aheavy atom unnatural amino acid, for example, can be useful for phasingx-ray structure data. The site-specific introduction of heavy atomsusing unnatural amino acids also provides selectivity and flexibility inchoosing positions for heavy atoms. Photoreactive unnatural amino acids(e.g., amino acids with benzophenone and arylazides (e.g., phenylazide)side chains), for example, allow for efficient in vivo and in vitrophotocrosslinking of proteins. Examples of photoreactive unnatural aminoacids include, but are not limited to, e.g., p-azido-phenylalanine andp-benzoyl-phenylalanine. The protein with the photoreactive unnaturalamino acids can then be crosslinked at will by excitation of thephotoreactive group-providing temporal (and/or spatial) control. In oneexample, the methyl group of an unnatural amino can be substituted withan isotopically labeled, e.g., methyl group, as a probe of localstructure and dynamics, e.g., with the use of nuclear magnetic resonanceand vibrational spectroscopy. Alkynyl or azido functional groups, forexample, allow the selective modification of proteins with moleculesthrough a [3+2] cycloaddition reaction.

Chemical Synthesis of Unnatural Amino Acids

Many of the unnatural amino acids provided above are commerciallyavailable, e.g., from Sigma (USA) or Aldrich (Milwaukee, Wis., USA).Those that are not commercially available are optionally synthesized asprovided herein or as provided in various publications or using standardmethods known to those of skill in the art. For organic synthesistechniques, see, e.g., Organic Chemistry by Fessendon and Fessendon,(1982, Second Edition, Willard Grant Press, Boston Mass.); AdvancedOrganic Chemistry by March (Third Edition, 1985, Wiley and Sons, NewYork); and Advanced Organic Chemistry by Carey and Sundberg (ThirdEdition, Parts A and B, 1990, Plenum Press, New York). Additionalpublications describing the synthesis of unnatural amino acids include,e.g., WO 2002/085923 entitled “In vivo incorporation of Unnatural AminoAcids;” Matsoukas et al., (1995) J. Med. Chem., 38, 4660-4669; King, F.E. & Kidd, D. A. A. (1949) A New Synthesis of Glutamine and ofγ-Dipeptides of Glutamic Acid from Phthylated Intermediates. J. Chem.Soc., 3315-3319; Friedman, O. M. & Chatterrji, R. (1959) Synthesis ofDerivatives of Glutamine as Model Substrates for Anti-Tumor Agents. J.Am. Chem. Soc. 81, 3750-3752; Craig, J. C. et al. (1988) AbsoluteConfiguration of the Enantiomers of 7-Chloro-4[[4-(diethylamino)-1-methylbutyl]amino]quinoline (Chloroquine). J. Org.Chem. 53, 1167-1170; Azoulay, M., Vilmont, M. & Frappier, F. (1991)Glutamine analogues as Potential Antimalarials, Eur. J. Med. Chem. 26,201-5; Koskinen, A. M. P. & Rapoport, H. (1989) Synthesis of4-Substituted Prolines as Conformationally Constrained Amino AcidAnalogues. J. Org. Chem. 54, 1859-1866; Christie, B. D. & Rapoport, H.(1985) Synthesis of Optically Pure Pipecolates from L-Asparagine.Application to the Total Synthesis of (+)-Apovincamine through AminoAcid Decarbonylation and Iminium Ion Cyclization. J. Org. Chem.1989:1859-1866; Barton et al., (1987) Synthesis of Novel a-Amino-Acidsand Derivatives Using Radical Chemistry: Synthesis of L- andD-a-Amino-Adipic Acids, L-a-aminopimelic Acid and AppropriateUnsaturated Derivatives.Tetrahedron Lett. 43:4297-4308; and, Subasingheet al., (1992) Quisqualic acid analogues: synthesis of beta-heterocyclic2-aminopropanoic acid derivatives and their activity at a novelquisqualate-sensitized site. J. Med. Chem. 35:4602-7.

Cellular Uptake of Unnatural Amino Acids

Unnatural amino acid uptake by a vertebrate cell is one issue that istypically considered when designing and selecting unnatural amino acids,e.g., for incorporation into a protein. For example, the high chargedensity of α-amino acids suggests that these compounds are unlikely tobe cell permeable. Natural amino acids are taken up into the vertebratecell via a collection of protein-based transport systems. A rapid screencan be done which assesses which unnatural amino acids, if any, aretaken up by cells. See, e.g., the toxicity assays in, e.g., U.S.Provisional Patent Application No. 60/435,821 (now U.S. Publication No.20040198637) entitled “Protein Arrays,” filed on Dec. 22, 2002; and Liu,D. R. & Schultz, P. G. (1999) Progress toward the evolution of anorganism with an expanded genetic code. PNAS United States 96:4780-4785,each incorporated herein by reference. Although uptake is easilyanalyzed with various assays, an alternative to designing unnaturalamino acids that are amenable to cellular uptake pathways is to providebiosynthetic pathways to create amino acids in vivo.

Biosynthesis of Unnatural Amino Acids

Many biosynthetic pathways already exist in cells for the production ofamino acids and other compounds. While a biosynthetic method for aparticular unnatural amino acid may not exist in nature, e.g., in avertebrate cell, the invention provides such methods. For example,biosynthetic pathways for unnatural amino acids are optionally generatedin host cell by adding new enzymes or modifying existing host cellpathways. Additional new enzymes are optionally naturally occurringenzymes or artificially evolved enzymes. For example, the biosynthesisof p-aminophenylalanine (as presented in an example in WO 2002/085923entitled “In vivo incorporation of unnatural amino acids”) relies on theaddition of a combination of known enzymes from other organisms. Thegenes for these enzymes can be introduced into a vertebrate cell bytransforming the cell with a plasmid comprising the genes. The genes,when expressed in the cell, provide an enzymatic pathway to synthesizethe desired compound. Examples of the types of enzymes that areoptionally added are provided in the examples below. Additional enzymessequences are found, e.g., in Genbank. Artificially evolved enzymes arealso optionally added into a cell in the same manner. In this manner,the cellular machinery and resources of a cell are manipulated toproduce unnatural amino acids.

A variety of methods are available for producing novel enzymes for usein biosynthetic pathways or for evolution of existing pathways. Forexample, recursive recombination, e.g., as developed by Maxygen, Inc.(available on the world wide web at www.maxygen.com), is optionally usedto develop novel enzymes and pathways. See, e.g., Stemmer (1994), Rapidevolution of a protein in vitro by DNA shuffling, Nature 370(4):389-391;and, Stemmer, (1994), DNA shuffling by random fragmentation andreassembly: In vitro recombination for molecular evolution, Proc. Natl.Acad. Sci. USA., 91:10747-10751. Similarly DesignPath™, developed byGenencor (available on the world wide web at genencor.com) is optionallyused for metabolic pathway engineering, e.g., to engineer a pathway tocreate O-methyl-L-tyrosine in a cell. This technology reconstructsexisting pathways in host organisms using a combination of new genes,e.g., identified through functional genomics, and molecular evolutionand design. Diversa Corporation (available on the world wide web atdiversa.com) also provides technology for rapidly screening libraries ofgenes and gene pathways, e.g., to create new pathways.

Typically, the unnatural amino acid produced with an engineeredbiosynthetic pathway of the invention is produced in a concentrationsufficient for efficient protein biosynthesis, e.g., a natural cellularamount, but not to such a degree as to affect the concentration of theother amino acids or exhaust cellular resources. Typical concentrationsproduced in vivo in this manner are about 10 mM to about 0.05 mM. Once acell is transformed with a plasmid comprising the genes used to produceenzymes desired for a specific pathway and an unnatural amino acid isgenerated, in vivo selections are optionally used to further optimizethe production of the unnatural amino acid for both ribosomal proteinsynthesis and cell growth.

Polypeptides with Unnatural Amino Acids

Proteins or polypeptides of interest with at least one unnatural aminoacid are a feature of the invention. The invention also includespolypeptides or proteins with at least one unnatural amino acid producedusing the compositions and methods of the invention. An excipient (e.g.,a pharmaceutically acceptable excipient) can also be present with theprotein.

By producing proteins or polypeptides of interest with at least oneunnatural amino acid in vertebrate cells, proteins or polypeptides willtypically include vertebrate posttranslational modifications. In certainembodiments, a protein includes at least one unnatural amino acid and atleast one post-translational modification that is made in vivo by avertebrate cell, where the post-translational modification is not madeby a prokaryotic cell. For example, the post-translation modificationincludes, e.g., acetylation, acylation, lipid-modification,palmitoylation, palmitate addition, phosphorylation, glycolipid-linkagemodification, glycosylation, and the like. In one aspect, thepost-translational modification includes attachment of anoligosaccharide (e.g., (GlcNAc-Man)₂-Man-GlcNAc-GlcNAc)) to anasparagine by a GlcNAc-asparagine linkage. See also, Table 7, whichlists some examples of N-linked oligosaccharides of vertebrate proteins(additional residues can also be present, which are not shown). Inanother aspect, the post-translational modification includes attachmentof an oligosaccharide (e.g., Gal-GalNAc, Gal-GlcNAc, etc.) to a serineor threonine by a GalNAc-serine or GalNAc-threonine linkage, or aGlcNAc-serine or a GlcNAc-threonine linkage.

TABLE 7 EXAMPLES OF OLIGOSACCHARIDES THROUGH GlcNAc-LINKAGE Type BaseStructure High-mannose

Hybrid

Complex

Xylose

In yet another aspect, the post-translation modification includesproteolytic processing of precursors (e.g., calcitonin precursor,calcitonin gene-related peptide precursor, preproparathyroid hormone,preproinsulin, proinsulin, prepro-opiomelanocortin, pro-opiomelanocortinand the like), assembly into a multisubunit protein or macromolecularassembly, translation to another site in the cell (e.g., to organelles,such as the endoplasmic reticulum, the golgi apparatus, the nucleus,lysosomes, peroxisomes, mitochondria, chloroplasts, vacuoles, etc., orthrough the secretory pathway). In certain embodiments, the proteincomprises a secretion or localization sequence, an epitope tag, a FLAGtag, a polyhistidine tag, a GST fusion, or the like.

One advantage of an unnatural amino acid is that it presents additionalchemical moieties that can be used to add additional molecules. Thesemodifications can be made in vivo in a vertebrate cell, or in vitro.Thus, in certain embodiments, the post-translational modification isthrough the unnatural amino acid. For example, the post-translationalmodification can be through a nucleophilic-electrophilic reaction. Mostreactions currently used for the selective modification of proteinsinvolve covalent bond formation between nucleophilic and electrophilicreaction partners, e.g. the reaction of α-haloketones with histidine orcysteine side chains. Selectivity in these cases is determined by thenumber and accessibility of the nucleophilic residues in the protein. Inproteins of the invention, other more selective reactions can be used,such as the reaction of an unnatural keto-amino acid with hydrazides oraminooxy compounds, in vitro and in vivo. See, e.g., Cornish, et al.,(1996) Am. Chem. Sec., 118:8150-8151; Mahal, at al., (1997) Science276:1125-1128; Wang, et al., (2001) Science 292:498-500; Chin, at al.,(2002) Am. Chem. Soc. 124:9026-9027; Chin, at al., (2002) Proc. Natl.Acad. Sci., 99:11020-11024; Wang, et al., (2003) Proc. Natl. Acad. Sci.,100:56-61; Zhang, at al., (2003) Biochemistry. 42:6735-6746; and, Chin,at al., (2003) Science, in press. This allows the selective labeling ofvirtually any protein with a host of reagents including fluorophores,crosslinking agents, saccharide derivatives and cytotoxic molecules. Seealso, patent application U.S. Ser. No. 10/686,944 entitled “Glycoproteinsynthesis” filed Oct. 15, 2003. Post-translational modifications, e.g.,through an azido amino acid, can also made through the Staudingerligation (e.g., with triarylphosphine reagents). See, e.g., Kiick etal., (2002) Incorporation of azides into recombinant proteins forchemoselective modification by the Staudinger ligtation, PNAS 99:19-24.

This invention provides another highly efficient method for theselective modification of proteins, which involves the geneticincorporation of unnatural amino acids, e.g., containing an azide oralkynyl moiety into proteins in response to a selector codon. Theseamino acid side chains can then be modified by, e.g., a Huisgen [3+2]cycloaddition reaction (see, e.g., Padwa, A. in Comprehensive OrganicSynthesis. Vol. 4, (1991) Ed. Trost, B. M., Pergamon, Oxford, p.1069-1109; and, Huisgen, R. in 1,3-Dipolar Cycloaddition Chemistry,(1984) Ed. Padwa, A., Wiley, New York, p. 1-176) with, e.g., alkynyl orazide derivatives, respectively. See, e.g., FIG. 16. Because this methodinvolves a cycloaddition rather than a nucleophilic substitution,proteins can be modified with extremely high selectivity. This reactioncan be carried out at room temperature in aqueous conditions withexcellent regioselectivity (1,4>1,5) by the addition of catalyticamounts of Cu(I) salts to the reaction mixture. See, e.g., Tornoe, atal., (2002) Org. Chem. 67:3057-3064; and, Rostovtsev, et al., (2002)Angew. Chem. Int. Ed 41:2596-2599. Another method that can be used isthe ligand exchange on a bisarsenic compound with a tetracysteine motif,see, e.g., Griffin, et al., (1998) Science 281:269-272.

A molecule that can be added to a protein of the invention through afunctional group of a non-naturally encoded amino acid includesvirtually any molecule with complementary functional group. Suchmolecules include, but are not limited to, dyes, fluorophores,crosslinking agents, saccharide derivatives, polymers (e.g., derivativesof polyethylene glycol), photocrosslinkers, cytotoxic compounds,affinity labels, derivatives of biotin, resins, beads, a second proteinor polypeptide (or more), polynucleotide(s) (e.g., DNA, RNA, etc.),metal chelators, cofactors, fatty acids, carbohydrates, and the like.

In another aspect, the invention provides compositions including suchmolecules and methods of producing these molecules, e.g., polyethyleneglycol derivatives, where n is an integer between, e.g., 50 and 10,000,75 and 5,000, 100 and 2,000, 100 and 1,000, etc. In embodiment of theinvention, the polyethylene glycol has a molecular weight of, e.g.,about 5,000 to about 100,000 Da, about 20,000 to about 30,000, about40,000, or about 50,000 Da, about 20,000 to about 10,000 Da, etc.

Various compositions comprising these compounds, e.g., with proteins andcells, are also provided. In one aspect of the invention, a proteincomprising an azido dye (e.g., of chemical structure 4 or chemicalstructure 6), further includes at least one unnatural amino acid (e.g.,an alkynyl amino acid), where the azido dye is attached to the unnaturalamino acid through a [3+2] cycloaddition.

A vertebrate cell of the invention provides the ability to synthesizeproteins that comprise unnatural amino acids in large useful quantities.In one aspect, the composition optionally includes, e.g., at least 10micrograms, at least 50 micrograms, at least 75 micrograms, at least 100micrograms, at least 200 micrograms, at least 250 micrograms, at least500 micrograms, at least 1 milligram, at least 10 milligrams or more ofthe protein that comprises an unnatural amino acid, or an amount thatcan be achieved with in vivo protein production methods (details onrecombinant protein production and purification are provided herein). Inanother aspect, the protein is optionally present in the composition ata concentration of, e.g., at least 10 micrograms of protein per liter,at least 50 micrograms of protein per liter, at least 75 micrograms ofprotein per liter, at least 100 micrograms of protein per liter, atleast 200 micrograms of protein per liter, at least 250 micrograms ofprotein per liter, at least 500 micrograms of protein per liter, atleast 1 milligram of protein per liter, or at least 10 milligrams ofprotein per liter or more, in, e.g., a cell lysate, a buffer, apharmaceutical buffer, or other liquid suspension (e.g., in a volume of,e.g., anywhere from about 1 nl to about 100 L). The production of largequantities (e.g., greater that that typically possible with othermethods, e.g., in vitro translation) of a protein in a vertebrate cellincluding at least one unnatural amino acid is a feature of theinvention.

The incorporation of an unnatural amino acid can be done to, e.g.,tailor changes in protein structure and/or function, e.g., to changesize, acidity, nucleophilicity, hydrogen bonding, hydrophobicity,accessibility of protease target sites, target to a moiety (e.g., for aprotein array), etc. Proteins that include an unnatural amino acid canhave enhanced or even entirely new catalytic or physical properties. Forexample, the following properties are optionally modified by inclusionof an unnatural amino acid into a protein: toxicity, biodistribution,structural properties, spectroscopic properties, chemical and/orphotochemical properties, catalytic ability, half-life (e.g., serumhalf-life), ability to react with other molecules, e.g., covalently ornoncovalently, and the like. The compositions including proteins thatinclude at least one unnatural amino acid are useful for, e.g., noveltherapeutics, diagnostics, catalytic enzymes, industrial enzymes,binding proteins (e.g., antibodies), and e.g., the study of proteinstructure and function. See, e.g., Dougherty, (2000) Unnatural AminoAcids as Probes of Protein Structure and Function, Current Opinion inChemical Biology. 4:645-652.

In one aspect of the invention, a composition includes at least oneprotein with at least one, e.g., at least two, at least three, at leastfour, at least five, at least six, at least seven, at least eight, atleast nine, or at least ten or more unnatural amino acids. The unnaturalamino acids can be the same or different, e.g., there can be 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 or more different sites in the protein thatcomprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different unnaturalamino acids. In another aspect, a composition includes a protein with atleast one, but fewer than all, of a particular amino acid present in theprotein is substituted with the unnatural amino acid. For a givenprotein with more than one unnatural amino acid, the unnatural aminoacids can be identical or different (e.g., the protein can include twoor more different types of unnatural amino acids, or can include two ofthe same unnatural amino acid). For a given protein with more than twounnatural amino acids, the unnatural amino acids can be the same,different or a combination of a multiple unnatural amino acid of thesame kind with at least one different unnatural amino acid.

Essentially any protein (or portion thereof) that includes an unnaturalamino acid (and any corresponding coding nucleic acid, e.g., whichincludes one or more selector codons) can be produced using thecompositions and methods herein. No attempt is made to identify thehundreds of thousands of known proteins, any of which can be modified toinclude one or more unnatural amino acid, e.g., by tailoring anyavailable mutation methods to include one or more appropriate selectorcodon in a relevant translation system. Common sequence repositories forknown proteins include GenBank EMBL, DDBJ and the NCBI. Otherrepositories can easily be identified by searching the internet.

Typically, the proteins are, e.g., at least 60%, at least 70%, at least75%, at least 80%, at least 90%, at least 95%, or at least 99% or moreidentical to any available protein (e.g., a therapeutic protein, adiagnostic protein, an industrial enzyme, or portion thereof, and thelike), and they comprise one or more unnatural amino acid. Examples oftherapeutic, diagnostic, and other proteins that can be modified tocomprise one or more unnatural amino acids include, but are not limitedto, e.g., Alpha-1 antitrypsin, Angiostatin, Antihemolytic factor,antibodies (further details on antibodies are found below),Apolipoprotein, Apoprotein, Atrial natriuretic factor, Atrialnatriuretic polypeptide, Atrial peptides, C-X-C chemokines (e.g.,T39765, NAP-2, ENA-78, Gro-a, Gro-b, Gro-c, IP-10, GCP-2, NAP-4, SDF-1,PF4, MIG), Calcitonin, CC chemokines (e.g., Monocyte chemoattractantprotein-1, Monocyte chemoattractant protein-2, Monocyte chemoattractantprotein-3, Monocyte inflammatory protein-1 alpha, Monocyte inflammatoryprotein-1 beta, RANTES, I309, R83915, R91733, HCC1, T58847, D31065,T64262), CD40 ligand, C-kit Ligand, Collagen, Colony stimulating factor(CSF), Complement factor 5a, Complement inhibitor, Complement receptor1, cytokines, (e.g., epithelial Neutrophil Activating Peptide-78,GROα/MGSA, GROβ, GROγ, MIP-1α, MIP-1δ, MCP-1), Epidermal Growth Factor(EGF), Erythropoietin (“EPO”, representing a preferred target formodification by the incorporation of one or more unnatural amino acid),Exfoliating toxins A and B, Factor IX, Factor VII, Factor VIII, FactorX, Fibroblast Growth Factor (FGF), Fibrinogen, Fibronectin, G-CSF,GM-CSF, Glucocerebrosidase, Gonadotropin, growth factors, Hedgehogproteins (e.g., Sonic, Indian, Desert), Hemoglobin, Hepatocyte GrowthFactor (HGF), Hirudin, Human serum albumin, Insulin, Insulin-like GrowthFactor (IGF), interferons (e.g., IFN-α, IFN-β, IFN-γ), interleukins(e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, etc.), Keratinocyte Growth Factor (KGF), Lactoferrin,leukemia inhibitory factor, Luciferase, Neurturin, Neutrophil inhibitoryfactor (NIF), oncostatin M, Osteogenic protein, Parathyroid hormone,PD-ECSF, PDGF, peptide hormones (e.g., Human Growth Hormone),Pleiotropin, Protein A, Protein G, Pyrogenic exotoxins A, B, and C,Relaxin, Renin, SCF, Soluble complement receptor I, Soluble I-CAM 1,Soluble interleukin receptors (IL-1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12,13, 14, 15), Soluble TNF receptor, Somatomedin, Somatostatin,Somatotropin, Streptokinase, Superantigens, i.e., Staphylococcalenterotoxins (SEA, SEB, SEC1, SEC2, SEC3, SED, SEE), Superoxidedismutase (SOD), Toxic shock syndrome toxin (TSST-1), Thymosin alpha 1,Tissue plasminogen activator, Tumor necrosis factor beta (TNF beta),Tumor necrosis factor receptor (TNFR), Tumor necrosis factor-alpha (TNFalpha), Vascular Endothelial Growth Factor (VEGEF), Urokinase, and manyothers.

One class of proteins that can be made using the compositions andmethods for in vivo incorporation of unnatural amino acids describedherein includes transcriptional modulators or portions thereof. Exampletranscriptional modulators include genes and transcriptional modulatorproteins that modulate cell growth, differentiation, regulation, or thelike. Transcriptional modulators are found in prokaryotes, viruses, andeukaryotes, including fungi, plants, yeasts, insects, and animals,including mammals, providing a wide range of therapeutic targets. Itwill be appreciated that expression and transcriptional activatorsregulate transcription by many mechanisms, e.g., by binding toreceptors, stimulating a signal transduction cascade, regulatingexpression of transcription factors, binding to promoters and enhancers,binding to proteins that bind to promoters and enhancers, unwinding DNA,splicing pre-mRNA, polyadenylating RNA, and degrading RNA. For example,compositions of GAL4 protein or portion thereof in a vertebrate cell arealso a feature of the invention. Typically, the GAL4 protein or portionthereof comprises at least one unnatural amino acid. See also thesection herein entitled “Orthogonal aminoacyl-tRNA synthetases.”

One class of proteins of the invention (e.g., proteins with one or moreunnatural amino acids) include expression activators such as cytokines,inflammatory molecules, growth factors, their receptors, and oncogeneproducts, e.g., interleukins (e.g., IL-1, IL-2, IL-8, etc.),interferons, FGF, IGF-I, IGF-II, FGF, PDGF, TNF, TGF-α, TGF-β, EGF, KGF,SCF/c-Kit, CD40/CD40, VLA-4/VNCAM-1, ICAM-1/LFA-1, and hyalurin/CD44;signal transduction molecules and corresponding oncogene products, e.g.,Mos, Ras, Raf, and Met; and transcriptional activators and suppressors,e.g., p53, Tat, Fos, Myc, Jun, Myb, Rel, and steroid hormone receptorssuch as those for estrogen, progesterone, testosterone, aldosterone, theLDL receptor ligand and corticosterone.

Enzymes (e.g., industrial enzymes), or portions thereof with at leastone unnatural amino acid, are also provided by the invention. Examplesof enzymes include, but are not limited to, e.g., amidases, amino acidracemases, acylases, dehalogenases, dioxygenases, diarylpropaneperoxidases, epimerases, epoxide hydrolases, esterases, isomerases,kinases, glucose isomerases, glycosidases, glycosyl transferases,haloperoxidases, monooxygenases (e.g., p450s), lipases, ligninperoxidases, nitrile hydratases, nitrilases, proteases, phosphatases,subtilisins, transaminase, and nucleases.

Many of these proteins are commercially available (See, e.g., the SigmaBioSciences 2002 catalogue and price list), and the correspondingprotein sequences and genes and, typically, many variants thereof, arewell-known (see, e.g., Genbank). Any of them can be modified by theinsertion of one or more unnatural amino acid according to theinvention, e.g., to alter the protein with respect to one or moretherapeutic, diagnostic or enzymatic properties of interest. Examples oftherapeutically relevant properties include serum half-life, shelfhalf-life, stability, immunogenicity, therapeutic activity,detectability (e.g., by the inclusion of reporter groups (e.g., labelsor label binding sites) in the unnatural amino acids), reduction of LD₅₀or other side effects, ability to enter the body through the gastrictract (e.g., oral availability), or the like. Examples of diagnosticproperties include shelf half-life, stability, diagnostic activity,detectability, or the like. Examples of relevant enzymatic propertiesinclude shelf half-life, stability, enzymatic activity, productioncapability, or the like.

A variety of other proteins can also be modified to include one or moreunnatural amino acid of the invention. For example, the invention caninclude substituting one or more natural amino acids in one or morevaccine proteins with an unnatural amino acid, e.g., in proteins frominfectious fungi, e.g., Aspergillus, Candida species; bacteria,particularly E. coli, which serves a model for pathogenic bacteria, aswell as medically important bacteria such as Staphylococci (e.g.,aureus), or Streptococci (e.g., pneumoniae); protozoa such as sporozoa(e.g., Plasmodia), rhizopods (e.g., Entamoeba) and flagellates(Trypanosoma, Leishmanla, Trichomonas, Giardia, etc.); viruses such as(+) RNA viruses (examples include Poxviruses e.g., vaccinia;Picornaviruses, e.g. polio; Togaviruses, e.g., rubella; Flaviviruses,e.g., HCV; and Coronaviruses), (−) RNA viruses (e.g., Rhabdoviruses,e.g., VSV; Paramyxovimses, e.g., RSV; Orthomyxovimses, e.g., influenza;Bunyaviruses; and Arenaviruses), dsDNA viruses (Reoviruses, forexample), RNA to DNA viruses, i.e., Retrovinruses, e.g., HIV and HTLV,and certain DNA to RNA viruses such as Hepatitis B.

Agriculturally related proteins such as insect resistance proteins(e.g., the Cry proteins), starch and lipid production enzymes, plant andinsect toxins, toxin-resistance proteins, Mycotoxin detoxificationproteins, plant growth enzymes (e.g., Ribulose 1,5-BisphosphateCarboxylase/Oxygenase, “RUBISCO”), lipoxygenase (LOX), andPhosphoenolpyruvate (PEP) carboxylase are also suitable targets forunnatural amino acid modification.

The invention also provides methods for producing in a vertebrate cellat least one protein comprising at least one unnatural amino acid (andproteins produced by such methods). For example, a method includes:growing, in an appropriate medium, a vertebrate cell that comprises anucleic acid that comprises at least one selector codon and encodes theprotein. The vertebrate cell also comprises: an orthogonal tRNA (O-tRNA)that functions in the cell and recognizes the selector codon; and anorthogonal aminoacyl tRNA synthetase (O-RS) that preferentiallyaminoacylates the O-tRNA with the unnatural amino acid, and the mediumcomprises an unnatural amino acid.

In one embodiment, the method further includes incorporating into theprotein the unnatural amino acid, where the unnatural amino acidcomprises a first reactive group; and contacting the protein with amolecule (e.g., a dye, a polymer, e.g., a derivative of polyethyleneglycol, a photocrosslinker, a cytotoxic compound, an affinity label, aderivative of biotin, a resin, a second protein or polypeptide, a metalchelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide(e.g., DNA, RNA, etc.), and the like) that comprises a second reactivegroup. The first reactive group reacts with the second reactive group toattach the molecule to the unnatural amino acid through a [3+2]cycloaddition. In one embodiment, the first reactive group is an alkynylor azido moiety and the second reactive group is an azido or alkynylmoiety. For example, the first reactive group is the alkynyl moiety(e.g., in unnatural amino acid p-propargyloxyphenylalanine) and thesecond reactive group is the azido moiety. In another example, the firstreactive group is the azido moiety (e.g., in the unnatural amino acidp-azido-L-phenylalanine) and the second reactive group is the alkynylmoiety.

In one embodiment, the O-RS aminoacylates the O-tRNA with the unnaturalamino acid at least 50% as efficiently as does an O-RS having an aminoacid sequence, e.g., as set forth in SEQ ID NO.: 86 or 45. In anotherembodiment, the O-tRNA comprises, is processed from, or is encoded bySEQ ID NO.: 65 or 64, or a complementary polynucleotide sequencethereof. In yet another embodiment, the O-RS comprises an amino acid setforth in any one of SEQ ID NO.: 36-63 and/or 86.

The encoded protein can comprise, e.g., a therapeutic protein, adiagnostic protein, an industrial enzyme, or portion thereof.Optionally, the protein that is produced by the method is furthermodified through the unnatural amino acid. For example, the proteinproduced by the method is optionally modified by at least onepost-translational modification in vivo.

Methods of producing a screening or selecting transcriptional modulatorprotein are also provided (and screening or selecting transcriptionalmodulator proteins produced by such methods). For example, a methodincludes: selecting a first polynucleotide sequence, where thepolynucleotide sequence encodes a nucleic acid binding domain; andmutating the first polynucleotide sequence to include at least oneselector codon. This provides a screening or selecting polynucleotidesequence. The method also includes: selecting a second polynucleotidesequence, where the second polynucleotide sequence encodes atranscriptional activation domain; providing a construct that comprisesthe screening or selecting polynucleotide sequence operably linked tothe second polynucleotide sequence; and, introducing the construct, anunnatural amino acid, an orthogonal tRNA synthetase (O-RS) and anorthogonal tRNA (O-tRNA) into a cell. With these components, the O-RSpreferentially aminoacylates the O-tRNA with the unnatural amino acidand the O-tRNA recognizes the selector codon and incorporates theunnatural amino acid into the nucleic acid binding domain, in responseto the selector codon in the screening or selecting polynucleotidesequence, thereby providing the screening or selecting transcriptionalmodulator protein.

In certain embodiments, the protein or polypeptide of interest (orportion thereof) in the methods and/or compositions of the invention isencoded by a nucleic acid. Typically, the nucleic acid comprises atleast one selector codon, at least two selector codons, at least threeselector codons, at least four selector codons, at least five selectorcodons, at least six selector codons, at least seven selector codons, atleast eight selector codons, at least nine selector codons, ten or moreselector codons.

Genes coding for proteins or polypeptides of interest can be mutagenizedusing methods well-known to one of skill in the art and described hereinunder “Mutagenesis and Other Molecular Biology Techniques” to include,e.g., one or more selector codon for the incorporation of an unnaturalamino acid. For example, a nucleic acid for a protein of interest ismutagenized to include one or more selector codon, providing for theinsertion of the one or more unnatural amino acids. The inventionincludes any such variant, e.g., mutant, versions of any protein, e.g.,including at least one unnatural amino acid. Similarly, the inventionalso includes corresponding nucleic acids, i.e., any nucleic acid withone or more selector codon that encodes one or more unnatural aminoacid.

Purifying Recombinant Proteins Comprising Unnatural Amino Acids

Proteins of the invention, e.g., proteins comprising unnatural aminoacids, antibodies to proteins comprising unnatural amino acids, etc.,can be purified, either partially or substantially to homogeneity,according to standard procedures known to and used by those of skill inthe art. Accordingly, polypeptides of the invention can be recovered andpurified by any of a number of methods well known in the art, including,e.g., ammonium sulfate or ethanol precipitation, acid or baseextraction, column chromatography, affinity column chromatography, anionor cation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, hydroxylapatite chromatography,lectin chromatography, gel electrophoresis and the like. Proteinrefolding steps can be used, as desired, in making correctly foldedmature proteins. High performance liquid chromatography (HPLC), affinitychromatography or other suitable methods can be employed in finalpurification steps where high purity is desired. In one embodiment,antibodies made against unnatural amino acids (or proteins comprisingunnatural amino acids) are used as purification reagents, e.g., foraffinity-based purification of proteins comprising one or more unnaturalamino acid(s). Once purified, partially or to homogeneity, as desired,the polypeptides are optionally used e.g., as assay components,therapeutic reagents or as immunogens for antibody production.

In addition to other references noted herein, a variety ofpurification/protein folding methods are well known in the art,including, e.g., those set forth in R. Scopes, Protein Purification.Springer-Verlag, N.Y. (1982); Deutscher, Methods in Enzymology Vol. 182:Guide to Protein Purification, Academic Press, Inc. N.Y. (1990); Sandana(1997) Biosparation of Proteins. Academic Press, Inc.; Bollag et al.(1996) Protein Methods, 2nd Edition Wiley-Liss, NY; Walker (1996) TheProtein Protocols Handbook Humana Press, NJ, Harris and Angal (1990)Protein Purification Applications: A Practical Approach IRL Press atOxford, Oxford, England; Harris and Angal Protein Purification Methods:A Practical Approach IRL Press at Oxford, Oxford, England; Scopes (1993)Protein Purification: Principles and Practice 3rd Edition SpringerVerlag, NY; Janson and Ryden (1998) Protein Purification: Principles.High Resolution Methods and Applications. Second Edition Wiley-VCH, NY;and Walker (1998) Protein Protocols on CD-ROM Humana Press, NJ; and thereferences cited therein.

One advantage of producing a protein or polypeptide of interest with anunnatural amino acid in a vertebrate cell is that typically the proteinsor polypeptides will be folded in their native conformations. However,in certain embodiments of the invention, those of skill in the art willrecognize that, after synthesis, expression and/or purification,proteins can possess a conformation different from the desiredconformations of the relevant polypeptides. In one aspect of theinvention, the expressed protein is optionally denatured and thenrenatured. This is accomplished, e.g., by adding a chaperonin to theprotein or polypeptide of interest, and/or by solubilizing the proteinsin a chaotropic agent such as guanidine HCl, etc.

In general, it is occasionally desirable to denature and reduceexpressed polypeptides and then to cause the polypeptides to re-foldinto the preferred conformation. For example, guanidine, urea, DTT, DTE,and/or a chaperonin can be added to a translation product of interest.Methods of reducing, denaturing and renaturing proteins are well knownto those of skill in the art (see, the references above, and Debinski,et al. (1993) J. Biol. Chem., 268: 14065-14070; Kreitman and Pastan(1993) Bioconjug. Chem., 4: 581-585; and Buchner, et al., (1992) Anal.Biochem., 205: 263-270). Debinski, et al., for example, describe thedenaturation and reduction of inclusion body proteins in guanidine-DTE.The proteins can be refolded in a redox buffer containing, e.g.,oxidized glutathione and L-arginine. Refolding reagents can be flowed orotherwise moved into contact with the one or more polypeptide or otherexpression product, or vice-versa.

Antibodies

In one aspect, the invention provides antibodies to molecules of theinvention, e.g., synthetases, tRNA, and proteins comprising unnaturalamino acids. Antibodies to molecules of the invention are useful aspurification reagents, e.g., for purifying the molecules of theinvention. In addition, the antibodies can be used as indicator reagentsto indicate the presence of a synthetase, a tRNA, or protein comprisingan unnatural amino acid, e.g., to track the presence or location (e.g.,in vivo or in situ) of the molecule.

An antibody of the invention can be a protein comprising one or morepolypeptides substantially or partially encoded by immunoglobulin genesor fragments of immunoglobulin genes. The recognized immunoglobulingenes include the kappa, lambda, alpha, gamma, delta, epsilon and muconstant region genes, as well as myriad immunoglobulin variable regiongenes. Light chains are classified as either kappa or lambda. Heavychains are classified as gamma, mu, alpha, delta, or epsilon, which inturn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,respectively. A typical immunoglobulin (e.g., antibody) structural unitcomprises a tetramer. Each tetramer is composed of two identical pairsof polypeptide chains, each pair having one “light” (about 25 kD) andone “heavy” chain (about 50-70 kD). The N-terminus of each chain definesa variable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain (VL)and variable heavy chain (V_(H)) refer to these light and heavy chains,respectively.

Antibodies exist as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab′)₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab′)₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region thereby converting theF(ab′)₂dimer into an Fab′ monomer. The Fab′ monomer is essentially anFab with part of the hinge region (see, Fundamental Immunology 4^(th)addition, W. E. Paul, ed., Raven Press, N.Y. (1999), for a more detaileddescription of other antibody fragments). While various antibodyfragments are defined in terms of the digestion of an intact antibody,one of skill will appreciate that such Fab′ fragments, etc. may besynthesized de novo either chemically or by utilizing recombinant DNAmethodology. Thus, the term antibody, as used herein, also optionallyincludes antibody fragments either produced by the modification of wholeantibodies or synthesized de novo using recombinant DNA methodologies.Antibodies include single chain antibodies, including single chain Fv(sFv or scFv) antibodies in which a variable heavy and a variable lightchain are joined together (directly or through a peptide linker) to forma continuous polypeptide. Antibodies of the invention can be, e.g.,polyclonal, monoclonal, chimeric, humanized, single chain, Fabfragments, fragments produced by an Fab expression library, or the like.

In general, antibodies of the invention are valuable, both as generalreagents and as therapeutic reagents in a variety of molecularbiological or pharmaceutical processes. Methods of producing polyclonaland monoclonal antibodies are available, and can be applied to makingthe antibodies of the invention. A number of basic texts describestandard antibody production processes, including, e.g., Borrebaeck (ed)(1995) Antibody Engineering, 2^(nd) Edition Freeman and Company, NY(Borrebaeck); McCafferty et al. (1996) Antibody Engineering. A PracticalApproach IRL at Oxford Press, Oxford, England (McCafferty), and Paul(1995) Antibody Engineering Protocols Humana Press, Towata, NJ (Paul);Paul (ed.), (1999) Fundamental Immunology, Fifth edition Raven Press,N.Y.; Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY;Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold SpringHarbor Press, NY; Stites et al. (eds.) Basic and Clinical Immunology(4th ed.) Lange Medical Publications, Los Altos, Calif., and referencescited therein; Goding (1986) Monoclonal Antibodies: Principles andPractice (2d ed.) Academic Press, New York, N.Y.; and Kohler andMilstein (1975) Nature 256: 495-497.

A variety of recombinant techniques for antibody preparation which donot rely on, e.g., injection of an antigen into an animal have beendeveloped and can be used in the context of the present invention. Forexample, it is possible to generate and select libraries of recombinantantibodies in phage or similar vectors. See, e.g., Winter et al. (1994)Making Antibodies by Phage Display Technology Annu. Rev. Immunol,12:433-55 and the references cited therein for a review. See also,Griffiths and Duncan (1998) Strategies for selection of antibodies byphage display Curr Opin Biotechnol 9: 102-8; Hoogenboom et al. (1998)Antibody phage display technology and its applications Immunotechnology4: 1-20; Gram et al. (1992) in vitro selection and affinity maturationof antibodies from a naïve combinatorial immunoglobulin library PNAS89:3576-3580; Huse et al. (1989) Science 246: 1275-1281; and Ward, etal. (1989) Nature 341: 544-546.

In one embodiment, antibody libraries can include repertoires of V genes(e.g., harvested from populations of lymphocytes or assembled in vitro)which are cloned for display of associated heavy and light chainvariable domains on the surface of filamentous bacteriophage. Phage areselected by binding to an antigen. Soluble antibodies are expressed fromphage infected bacteria and the antibody can be improved, e.g., viamutagenesis. See e.g., Balint and Larrick (1993) Antibody Engineering byParsimonious Mutagenesis Gene 137:109-118; Stemmer et al. (1993)Selection of an Active Single Chain Fv Antibody From a Protein LinkerLibrary Prepared by Enzymatic Inverse PCR Biotechniques 14(2):256-65;Crameri et al. (1996) Construction and evolution of antibody-phagelibraries by DNA shuffling Nature Medicine 2:100-103; and Crameri andStemmer (1995) Combinatorial multiple cassette mutagenesis creates allthe permutations of mutant and wildtype cassettes Biotechniques18:194-195.

Kits for cloning and expression of recombinant antibody phage systemsare also known and available, e.g., the “recombinant phage antibodysystem, mouse ScFv module,” from Amersham-Pharmacia Biotechnology(Uppsala, Sweden). Bacteriophage antibody libraries have also beenproduced for making high affinity human antibodies by chain shuffling(See, e.g., Marks et al. (1992) By-Passing Immunization: Building HighAffinity Human Antibodies by Chain Shuffling Biotechniques 10:779-782.It will also be recognized that antibodies can be prepared by any of anumber of commercial services (e.g., Bethyl Laboratories (Montgomery,Tex.), Anawa (Switzerland), Eurogentec (Belgium and in the US inPhiladelphia, Pa., etc.) and many others.

In certain embodiments, it is useful to “humanize” antibodies of theinvention, e.g., where the antibodies are to be administeredtherapeutically. The use of humanized antibodies tends to reduce theincidence of unwanted immune responses against the therapeuticantibodies (e.g., when the patient is a human). The antibody referencesabove describe humanization strategies. In addition to humanizedantibodies, human antibodies are also a feature of the invention. Humanantibodies consist of characteristically human immunoglobulin sequences.Human antibodies can be produced in using a wide variety of methods(see, e.g., Larrick et al., U.S. Pat. No. 5,001,065, for a review). Ageneral approach for producing human antibodies by trioma technology isdescribed by Ostberg et al. (1983), Hybridoma 2: 361-367, Ostberg, U.S.Pat. No. 4,634,664, and Engelman et al., U.S. Pat. No. 4,634,666.

A variety of methods of using antibodies in the purification anddetection of proteins are known and can be applied to detecting andpurifying proteins comprising unnatural amino acids as noted herein. Ingeneral, antibodies are useful reagents for ELISA, western blotting,immunochemistry, affinity chromatograpy methods, SPR, and many othermethods. The references noted above provide details on how to performELISA assays, western blots, surface plasmon resonance (SPR) and thelike.

In one aspect of the invention, antibodies of the invention themselvesinclude unnatural amino acids, providing the antibodies with propertiesof interest (e.g., improved half-life, stability, toxicity, or thelike). See also, the section herein entitled “Polypeptides withunnatural amino acids.” Antibodies account for nearly 50% of allcompounds currently in clinical trials (Wittrup, (1999) Phage on displayTibtech 17: 423-424 and antibodies are used ubiquitously as diagnosticreagents. Accordingly, the ability to modify antibodies with unnaturalamino acids provides an important tool for modifying these valuablereagents.

For example, there are many applications of MAbs to the field ofdiagnostics. Assays range from simple spot tests to more involvedmethods such as the radio-labeled NR-LU-10 MAb from DuPont Merck Co.used for tumor imaging (Rusch et al. (1993) NR-LU-10 monoclonal antibodyscanning. A helpful new adjunct to computed tomography in evaluatingnon-small-cell lung cancer. J Thorac Cardiovasc Surg 106: 200-4). Asnoted, MAbs are central reagents for ELISA, western blotting,immunochemistry, affinity chromatograpy methods and the like. Any suchdiagnostic antibody can be modified to include one or more unnaturalamino acid, altering, e.g., the specificity or avidity of the Ab for atarget, or altering one or more detectable property, e.g., by includinga detectable label (e.g., spectrographic, fluorescent, luminescent,etc.) in the unnatural amino acid.

One class of valuable antibody reagents are therapeutic Abs. Forexample, antibodies can be tumor-specific MAbs that arrest tumor growthby targeting tumor cells for destruction by antibody-dependentcell-mediated cytotoxicity (ADCC) or complement-mediated lysis (CML)(these general types of Abs are sometimes referred to as “magicbullets”). One example is Rituxan, an anti-CD20 MAb for the treatment ofNon-Hodgkins lymphoma (Scott (1998) Rituximab: a new therapeuticmonoclonal antibody for non-Hodgkin's lymphoma Cancer Pract 6: 195-7). Asecond example relates to antibodies which interfere with a criticalcomponent of tumor growth. Herceptin is an anti-HER-2 monoclonalantibody for treatment of metastatic breast cancer, and provides anexample of an antibody with this mechanism of action (Baselga et al.(1998) Recombinant humanized anti-HER2 antibody (Herceptin) enhances theantitumor activity of paclitaxel and doxorubicin against HER2/neuoverexpressing human breast cancer xenografts [published erratum appearsin Cancer Res (1999) 59(8):2020], Cancer Res 58: 2825-31). A thirdexample relates to antibodies for delivery of cytotoxic compounds(toxins, radionuclides, etc.) directly to a tumor or other site ofinterest. For example, one application Mab is CYT-356, a 90Y-linkedantibody that targets radiation directly to prostate tumor cells (Deb etal. (1996) Treatment of hormone-refractory prostate cancer with90Y-CYT-356 monoclonal antibody Clin Cancer Res 2: 1289-97. A fourthapplication is antibody-directed enzyme prodrug therapy, where an enzymeco-localized to a tumor activates a systemically-administered pro-drugin the tumor vicinity. For example, an anti-Ep-CAM1 antibody linked tocarboxypeptidase A is being developed for treatment of colorectal cancer(Wolfe et al. (1999) Antibody-directed enzyme prodrug therapy with theT268G mutant of human carboxypeptidase A1: in vitro and in vivo studieswith prodrugs of methotrexate and the thymidylate synthase inhibitorsGW1031 and GW1843 Bioconjug Chem 10: 38-48). Other Abs (e.g.,antagonists) are designed to specifically inhibit normal cellularfunctions for therapeutic benefit. An example is Orthoclone OKT3, ananti-CD3 MAb offered by Johnson and Johnson for reducing acute organtransplant rejection (Strate et al. (1990) Orthoclone OKT3 as first-linetherapy in acute renal allograft rejection Transplant Proc 22: 219-20.Another class of antibody products are agonists. These Mabs are designedto specifically enhance normal cellular functions for therapeuticbenefit. For example, Mab-based agonists of acetylcholine receptors forneurotherapy are under development (Xie et al. (1997) Directdemonstration of MuSK involvement in acetylcholine receptor clusteringthrough identification of agonist ScFv Nat. Biotechnol. 15: 768-71. Anyof these antibodies can be modified to include one or more unnaturalamino acid to enhance one or more therapeutic property (specificity,avidity, serum-half-life, etc.).

Another class of antibody products provide novel functions. The mainantibodies in this group are catalytic antibodies such as Ig sequencesthat have been engineered to mimic the catalytic abilities of enzymes(Wentworth and Janda (1998) Catalytic antibodies Curr Opin Chem Biol 2:138-44. For example, an interesting application involves using thecatalytic antibody mAb-15A10 to hydrolyze cocaine in vive for addictiontherapy (Mets et al. (1998) A catalytic antibody against cocaineprevents cocaine's reinforcing and toxic effects in rats Proc Natl AcadSci USA 95: 10176-81). Catalytic antibodies can also be modified toinclude one or more unnatural amino acid to improve one or more propertyof interest.

Defining Polypeptides by Immunoreactivity

Because the polypeptides of the invention provide a variety of newpolypeptide sequences (e.g., comprising unnatural amino acids in thecase of proteins synthesized in the translation systems herein, or,e.g., in the case of the novel synthetases herein, novel sequences ofstandard amino acids), the polypeptides also provide new structuralfeatures which can be recognized, e.g., in immunological assays. Thegeneration of antibodies or antibodies which specifically bind thepolypeptides of the invention, as well as the polypeptides which arebound by such antibodies or antisera, are a feature of the invention.

For example, the invention includes synthetase proteins thatspecifically bind to or that are specifically immunoreactive with anantibody or antisera generated against an immunogen comprising an aminoacid sequence selected from one or more of (SEQ ID NO: 36-63, and/or86). To eliminate cross-reactivity with other homologues, the antibodyor antisera is subtracted with available control synthetase homologues,such as the wild-type E. coli tyrosyl synthetase (TyrRS) (e.g., SEQ IDNO.:2).

In one typical format, the immunoassay uses a polyclonal antiserum whichwas raised against one or more polypeptide comprising one or more of thesequences corresponding to one or more of SEQ ID NO: 36-63, and/or 86,or a substantial subsequence thereof (i.e., at least about 30% of thefull length sequence provided). The set of potential polypeptideimmunogens derived from SEQ ID NO: 36-63 and 86 are collectivelyreferred to below as “the immunogenic polypeptides.” The resultingantisera is optionally selected to have low cross-reactivity against thecontrol synthetase homologues and any such cross-reactivity is removed,e.g., by immunoabsorption, with one or more control synthetasehomologues, prior to use of the polyclonal antiserum in the immunoassay.

In order to produce antisera for use in an immunoassay, one or more ofthe immunogenic polypeptides is produced and purified as describedherein. For example, recombinant protein can be produced in arecombinant cell. An inbred strain of mice (used in this assay becauseresults are more reproducible due to the virtual genetic identity of themice) is immunized with the immunogenic protein(s) in combination with astandard adjuvant, such as Freund's adjuvant, and a standard mouseimmunization protocol (see, e.g., Harlow and Lane (1988) Antibodies. ALaboratory Manual, Cold Spring Harbor Publications, New York, for astandard description of antibody generation, immunoassay formats andconditions that can be used to determine specific immunoreactivity.Additional references and discussion of antibodies is also found hereinand can be applied here to make antibodies that define/detectpolypeptides by immunoreactivity). Alternatively, one or more syntheticor recombinant polypeptide derived from the sequences disclosed hereinis conjugated to a carrier protein and used as an immunogen.

Polyclonal sera are collected and titered against the immunogenicpolypeptide in an immunoassay, for example, a solid phase immunoassaywith one or more of the immunogenic proteins immobilized on a solidsupport. Polyclonal antisera with a titer of 10⁶ or greater areselected, pooled and subtracted with the control synthetase polypeptidesto produce subtracted pooled titered polyclonal antisera.

The subtracted pooled titered polyclonal antisera are tested for crossreactivity against the control homologues in a comparative immunoassay.In this comparative assay, discriminatory binding conditions aredetermined for the subtracted titered polyclonal antisera which resultin at least about a 5-10 fold higher signal to noise ratio for bindingof the titered polyclonal antisera to the immunogenic synthetase ascompared to binding to a control synthetase homologue. That is, thestringency of the binding/washing reaction(s) is/are adjusted by theaddition of non-specific competitors such as albumin or non-fat drymilk, and/or by adjusting salt conditions, temperature, and/or the like.These binding/washing conditions are used in subsequent assays fordetermining whether a test polypeptide (a polypeptide being compared tothe immunogenic polypeptides and/or the control polypeptides) isspecifically bound by the pooled subtracted polyclonal antisera. Inparticular, test polypeptides which show at least a 2-5× higher signalto noise ratio than the control synthetase homologue underdiscriminatory binding conditions, and at least about a ½ signal tonoise ratio as compared to the immunogenic polypeptide(s), sharessubstantial structural similarity with the immunogenic polypeptide ascompared to known synthetases, and is, therefore a polypeptide of theinvention.

In another example, immunoassays in the competitive binding format areused for detection of a test polypeptide. For example, as noted,cross-reacting antibodies are removed from the pooled antisera mixtureby immunoabsorbtion with the control polypeptides. The immunogenicpolypeptide(s) are then immobilized to a solid support which is exposedto the subtracted pooled antisera. Test proteins are added to the assayto compete for binding to the pooled subtracted antisera. The ability ofthe test protein(s) to compete for binding to the pooled subtractedantisera as compared to the immobilized protein(s) is compared to theability of the immunogenic polypeptide(s) added to the assay to competefor binding (the immunogenic polypeptides compete effectively with theimmobilized immunogenic polypeptides for binding to the pooledantisera). The percent cross-reactivity for the test proteins iscalculated, using standard calculations.

In a parallel assay, the ability of the control proteins to compete forbinding to the pooled subtracted antisera is optionally determined ascompared to the ability of the immunogenic polypeptide(s) to compete forbinding to the antisera. Again, the percent cross-reactivity for thecontrol polypeptides is calculated, using standard calculations. Wherethe percent cross-reactivity is at least 5-10× as high for the testpolypeptides as compared to the control polypeptides and or where thebinding of the test polypeptides is approximately in the range of thebinding of the immunogenic polypeptides, the test polypeptides are saidto specifically bind the pooled subtracted antisera.

In general, the immunoabsorbed and pooled antisera can be used in acompetitive binding immunoassay as described herein to compare any testpolypeptide to the immunogenic and/or control polypeptide(s). In orderto make this comparison, the immunogenic, test and control polypeptidesare each assayed at a wide range of concentrations and the amount ofeach polypeptide required to inhibit 50% of the binding of thesubtracted antisera to, e.g., an immobilized control, test orimmunogenic protein is determined using standard techniques. If theamount of the test polypeptide required for binding in the competitiveassay is less than twice the amount of the immunogenic polypeptide thatis required, then the test polypeptide is said to specifically bind toan antibody generated to the immunogenic protein, provided the amount isat least about 5-10× as high as for the control polypeptide.

As an additional determination of specificity, the pooled antisera isoptionally fully immunosorbed with the immunogenic polypeptide(s)(rather than the control polypeptides) until little or no binding of theresulting immunogenic polypeptide subtracted pooled antisera to theimmunogenic polypeptide(s) used in the immunoabsorption is detectable.This fully immunosorbed antisera is then tested for reactivity with thetest polypeptide. If little or no reactivity is observed (i.e., no morethan 2× the signal to noise ratio observed for binding of the fullyimmunosorbed antisera to the immunogenic polypeptide), then the testpolypeptide is specifically bound by the antisera elicited by theimmunogenic protein.

Pharmaceutical Compositions

The polypeptides or proteins of the invention (e.g., synthetases,proteins comprising one or more unnatural amino acid, etc.) areoptionally employed for therapeutic uses, e.g., in combination with asuitable pharmaceutical carrier. Such compositions, e.g., comprise atherapeutically effective amount of the compound, and a pharmaceuticallyacceptable carrier or excipient. Such a carrier or excipient includes,but is not limited to, saline, buffered saline, dextrose, water,glycerol, ethanol, and/or combinations thereof. The formulation is madeto suit the mode of administration. In general, methods of administeringproteins are well known in the art and can be applied to administrationof the polypeptides of the invention.

Therapeutic compositions comprising one or more polypeptide of theinvention are optionally tested in one or more appropriate in vitroand/or in vivo animal models of disease, to confirm efficacy, tissuemetabolism, and to estimate dosages, according to methods well known inthe art. In particular, dosages can be initially determined by activity,stability or other suitable measures of unnatural herein to naturalamino acid homologues (e.g., comparison of an EPO modified to includeone or more unnatural amino acids to a natural amino acid EPO), i.e., ina relevant assay.

Administration is by any of the routes normally used for introducing amolecule into ultimate contact with blood or tissue cells. The unnaturalamino acid polypeptides of the invention are administered in anysuitable manner, optionally with one or more pharmaceutically acceptablecarriers. Suitable methods of administering such polypeptides in thecontext of the present invention to a patient are available, and,although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective action or reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention.

Polypeptide compositions can be administered by a number of routesincluding, but not limited to: oral, intravenous, intraperitoneal,intramuscular, transdermal, subcutaneous, topical, sublingual, or rectalmeans. Unnatural amino acid polypeptide compositions can also beadministered via liposomes. Such administration routes and appropriateformulations are generally known to those of skill in the art.

The unnatural amino acid polypeptide, alone or in combination with othersuitable components, can also be made into aerosol formulations (i.e.,they can be “nebulized”) to be administered via inhalation. Aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations of packaged nucleic acid can be presented in unit-doseor multi-dose sealed containers, such as ampules and vials.

Parenteral administration and intravenous administration are preferredmethods of administration. In particular, the routes of administrationalready in use for natural amino acid homologue therapeutics (e.g.,those typically used for EPO, GCSF, GMCSF, IFNs, interleukins,antibodies, and/or any other pharmaceutically delivered protein), alongwith formulations in current use, provide preferred routes ofadministration and formulation for the proteins that include unnaturalamino acids of the invention (e.g., pegylated variants of currentthereputic proteins, etc.).

The dose administered to a patient, in the context of the presentinvention, is sufficient to effect a beneficial therapeutic response inthe patient over time, or, e.g., to inhibit infection by a pathogen, orother appropriate activity, depending on the application. The dose isdetermined by the efficacy of a particular composition/formulation, andthe activity, stability or serum half-life of the unnatural amino acidpolypeptide employed and the condition of the patient, as well as thebody weight or surface area of the patient to be treated. The size ofthe dose is also determined by the existence, nature, and extent of anyadverse side-effects that accompany the administration of a particularcomposition/formulation, or the like in a particular patient.

In determining the effective amount of the composition/formulation to beadministered in the treatment or prophylaxis of disease (e.g., cancers,inherited diseases, diabetes, AIDS, or the like), the physicianevaluates circulating plasma levels, formulation toxicities, progressionof the disease, and/or where relevant, the production of anti-unnaturalamino acid polypeptide antibodies.

The dose administered, e.g., to a 70 kilogram patient, is typically inthe range equivalent to dosages of currently-used therapeutic proteins,adjusted for the altered activity or serum half-life of the relevantcomposition. The compositions/formulations of this invention cansupplement treatment conditions by any known conventional therapy,including antibody administration, vaccine administration,administration of cytotoxic agents, natural amino acid polypeptides,nucleic acids, nucleotide analogues, biologic response modifiers, andthe like.

For administration, formulations of the present invention areadministered at a rate determined by the LD-50 of the relevantformulation, and/or observation of any side-effects of the unnaturalamino acids at various concentrations, e.g., as applied to the mass andoverall health of the patient. Administration can be accomplished viasingle or divided doses.

If a patient undergoing infusion of a formulation develops fevers,chills, or muscle aches, he/she receives the appropriate dose ofaspirin, ibuprofen, acetaminophen or other pain/fever controlling drug.Patients who experience reactions to the infusion such as fever, muscleaches, and chills are premedicated 30 minutes prior to the futureinfusions with either aspirin, acetaminophen, or, e.g., diphenhydramine.Meperidine is used for more severe chills and muscle aches that do notquickly respond to antipyretics and antihistamines. Treatment is slowedor discontinued depending upon the severity of the reaction.

Nucleic Acid and Polypeptide Sequence and Variants

As described above and below, the invention provides for nucleic acidpolynucleotide sequences and polypeptide amino acid sequences, e.g.,O-tRNA's and O-RSs, and, e.g., compositions and methods comprising saidsequences. Examples of said sequences, e.g., O-tRNA's and O-RSs aredisclosed herein (see, Table 5, e.g., SEQ ID NO. 3-65, 86, and otherthan SEQ ID NO.: 1 and 2). However, one of skill in the art willappreciate that the invention is not limited to those sequencesdisclosed herein, e.g., the Examples and Table 5. One of skill willappreciate that the invention also provides many related and evenunrelated sequences with the functions described herein, e.g., encodingan O-tRNA or an O-RS.

The invention also provides polypeptides (O-RSs) and polynucleotides,e.g., O-tRNA, polynucleotides that encode O-RSs or portions thereof(e.g., the active site of the synthetase), oligonucleotides used toconstruct aminoacyl-tRNA synthetase mutants, etc. For example, apolypeptide of the invention includes a polypeptide that comprises anamino acid sequence as shown in any one of SEQ ID NO.: 36-63, and/or 86,a polypeptide that comprises an amino acid sequence encoded by apolynucleotide sequence as shown in any one of SEQ ID NO.: 3-35, and apolypeptide that is specifically immunoreactive with an antibodyspecific for a polypeptide that comprises an amino acid sequence asshown in any one of SEQ ID NO.: 36-63, and/or 86, or a polypeptide thatcomprises an amino acid sequence encoded by a polynucleotide sequence asshown in any one of SEQ ID NO.: 3-35.

Also included among the polypeptides of the invention are polypeptidesthat comprise an amino acid sequence that is at least 90% identical tothat of a naturally occurring tyrosyl aminoacyl-tRNA synthetase (TyrRS)(e.g., SEQ ID NO.:2) and comprises two or more amino acids of groupsA-E. For example, group A includes valine, isoleucine, leucine, glycine,serine, alanine, or threonine at a position corresponding to Tyr37 of E.coli TyrRS; group B includes aspartate at a position corresponding toAsn126 of E. coli TyrRS; group C includes threonine, serine, arginine,asparagine or glycine at a position corresponding to Asp182 of E. coliTyrRS; group D includes methionine, alanine, valine, or tyrosine at aposition corresponding to Phe183 of E. coli TyrRS; and, group E includesserine, methionine, valine, cysteine, threonine, or alanine at aposition corresponding to Leu186 of E. coli TyrRS. Similarly,polypeptides of the invention also include a polypeptide that comprisesat least 20 contiguous amino acids of SEQ ID NO.: 36-63, and/or 86, andtwo or more amino acid substitutions as indicated above in groups A-E.An amino acid sequence comprising a conservative variation of any of theabove polypeptides is also included as a polypeptide of the invention.

In one embodiment, a composition includes a polypeptide of the inventionand an excipient (e.g., buffer, water, pharmaceutically acceptableexcipient, etc.). The invention also provides an antibody or antiseraspecifically immunoreactive with a polypeptide of the invention.

Polynucleotides are also provided in the invention. Polynucleotides ofthe invention include those that encode proteins or polypeptides ofinterest of the invention, or that include one or more selector codon,or both. For example, polynucleotides of the invention include, e.g., apolynucleotide comprising a nucleotide sequence as set forth in any oneof SEQ ID NO.: 3-35, 64-85; a polynucleotide that is complementary to orthat encodes a polynucleotide sequence thereof; and/or a polynucleotideencoding a polypeptide that comprises an amino acid sequence as setforth in any one of SEQ ID NO.: 36-63, and/or 86, or a conservativevariation thereof. A polynucleotide of the invention also includes apolynucleotide that encodes a polypeptide of the invention. Similarly, anucleic acid that hybridizes to a polynucleotide indicated above underhighly stringent conditions over substantially the entire length of thenucleic acid is a polynucleotide of the invention.

A polynucleotide of the invention also includes a polynucleotide thatencodes a polypeptide that comprises an amino acid sequence that is atleast 90% identical to that of a naturally occurring tyrosylaminoacyl-tRNA synthetase (TyrRS) (e.g., SEQ ID NO.: 2) and comprisestwo or more mutations as indicated above in groups A-E in paragraph 11.A polynucleotide that is that is at least 70%, (or at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, orleast 99% or more) identical to a polynucleotide indicated above and/ora polynucleotide comprising a conservative variation of any of thepolynucleotides indicated above are also included among thepolynucleotides of the invention.

In certain embodiments, a vector (e.g., a plasmid, a cosmid, a phage, avirus, etc.) comprises a polynucleotide of the invention. In oneembodiment, the vector is an expression vector. In another embodiment,the expression vector includes a promoter operably linked to one or moreof the polynucleotides of the invention. In another embodiment, a cellcomprises a vector that includes a polynucleotide of the invention.

One of skill will also appreciate that many variants of the disclosedsequences are included in the invention. For example, conservativevariations of the disclosed sequences that yield a functionallyidentical sequence are included in the invention. Variants of thenucleic acid polynucleotide sequences, wherein the variants hybridize toat least one disclosed sequence, are considered to be included in theinvention. Unique subsequences of the sequences disclosed herein, asdetermined by, e.g., standard sequence comparison techniques, are alsoincluded in the invention.

Conservative Variations

Owing to the degeneracy of the genetic code, “silent substitutions”(i.e., substitutions in a nucleic acid sequence which do not result inan alteration in an encoded polypeptide) are an implied feature of everynucleic acid sequence which encodes an amino acid. Similarly,“conservative amino acid substitutions,” in one or a few amino acids inan amino acid sequence are substituted with different amino acids withhighly similar properties, are also readily identified as being highlysimilar to a disclosed construct. Such conservative variations of eachdisclosed sequence are a feature of the present invention.

“Conservative variations” of a particular nucleic acid sequence refersto those nucleic acids which encode identical or essentially identicalamino acid sequences, or, where the nucleic acid does not encode anamino acid sequence, to essentially identical sequences. One of skillwill recognize that individual substitutions, deletions or additionswhich alter, add or delete a single amino acid or a small percentage ofamino acids (typically less than 5%, more typically less than 4%, 2% or1%) in an encoded sequence are “conservatively modified variations”where the alterations result in the deletion of an amino acid, additionof an amino acid, or substitution of an amino acid with a chemicallysimilar amino acid. Thus, “conservative variations” of a listedpolypeptide sequence of the present invention include substitutions of asmall percentage, typically less than 5%, more typically less than 2% or1%, of the amino acids of the polypeptide sequence, with aconservatively selected amino acid of the same conservative substitutiongroup. Finally, the addition of sequences that do not alter the encodedactivity of a nucleic acid molecule, such as the addition of anon-functional sequence, is a conservative variation of the basicnucleic acid.

Conservative substitution tables providing functionally similar aminoacids are well known in the art. The following sets forth example groupswhich contain natural amino acids that include “conservativesubstitutions” for one another.

Conservative Substitution Groups

1 Alanine (A) Serine (S) Threonine (T) 2 Aspartic acid (D) Glutamic acid(E) 3 Asparagine (N) Glutamine (Q) 4 Arginine (R) Lysine (K) 5Isoleucine (I) Leucine (L) Methionine (M) Valine (V) 6 Phenylalanine (F)Tyrosine (Y) Tryptophan (W)

Nucleic Acid Hybridization

Comparative hybridization can be used to identify nucleic acids of theinvention, including conservative variations of nucleic acids of theinvention, and this comparative hybridization method is a preferredmethod of distinguishing nucleic acids of the invention. In addition,target nucleic acids which hybridize to the nucleic acids represented bySEQ ID NO: 3-35, 64-85 under high, ultra-high and ultra-ultra highstringency conditions are a feature of the invention. Examples of suchnucleic acids include those with one or a few silent or conservativenucleic acid substitutions as compared to a given nucleic acid sequence.

A test nucleic acid is said to specifically hybridize to a probe nucleicacid when it hybridizes at least ½ as well to the probe as to theperfectly matched complementary target, i.e., with a signal to noiseratio at lest ½ as high as hybridization of the probe to the targetunder conditions in which the perfectly matched probe binds to theperfectly matched complementary target with a signal to noise ratio thatis at least about 5×-10× as high as that observed for hybridization toany of the unmatched target nucleic acids.

Nucleic acids “hybridize” when they associate, typically in solution.Nucleic acids hybridize due to a variety of well characterizedphysico-chemical forces, such as hydrogen bonding, solvent exclusion,base stacking and the like. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes part I chapter 2, “Overview of principles of hybridization andthe strategy of nucleic acid probe assays,” (Elsevier, New York), aswell as in Ausubel, supra. Hames and Higgins (1995) Gene Probes 1 IRLPress at Oxford University Press, Oxford, England, (Hames and Higgins 1)and Hames and Higgins (1995) Gene Probes 2 IRL Press at OxfordUniversity Press, Oxford, England (Hames and Higgins 2) provide detailson the synthesis, labeling, detection and quantification of DNA and RNA,including oligonucleotides.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or northern blot is 50% formalin with1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of stringent wash conditions is a 0.2×SSC wash at65° C. for 15 minutes (see, Sambrook, supra for a description of SSCbuffer). Often the high stringency wash is preceded by a low stringencywash to remove background probe signal. An example low stringency washis 2×SSC at 40° C. for 15 minutes. In general, a signal to noise ratioof 5× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization.

“Stringent hybridization wash conditions” in the context of nucleic acidhybridization experiments such as Southern and northern hybridizationsare sequence dependent, and are different under different environmentalparameters. An extensive guide to the hybridization of nucleic acids isfound in Tijssen (1993), supra. and in Hames and Higgins, 1 and 2.Stringent hybridization and wash conditions can easily be determinedempirically for any test nucleic acid. For example, in determininghighly stringent hybridization and wash conditions, the hybridizationand wash conditions are gradually increased (e.g., by increasingtemperature, decreasing salt concentration, increasing detergentconcentration and/or increasing the concentration of organic solventssuch as formalin in the hybridization or wash), until a selected set ofcriteria are met. For example, the hybridization and wash conditions aregradually increased until a probe binds to a perfectly matchedcomplementary target with a signal to noise ratio that is at least 5× ashigh as that observed for hybridization of the probe to an unmatchedtarget.

“Very stringent” conditions are selected to be equal to the thermalmelting point (T_(m)) for a particular probe. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetest sequence hybridizes to a perfectly matched probe. For the purposesof the present invention, generally, “highly stringent” hybridizationand wash conditions are selected to be about 5° C. lower than the T_(m)for the specific sequence at a defined ionic strength and pH.

“Ultra high-stringency” hybridization and wash conditions are those inwhich the stringency of hybridization and wash conditions are increaseduntil the signal to noise ratio for binding of the probe to theperfectly matched complementary target nucleic acid is at least 10× ashigh as that observed for hybridization to any of the unmatched targetnucleic acids. A target nucleic acid which hybridizes to a probe undersuch conditions, with a signal to noise ratio of at least % that of theperfectly matched complementary target nucleic acid is said to bind tothe probe under ultra-high stringency conditions.

Similarly, even higher levels of stringency can be determined bygradually increasing the hybridization and/or wash conditions of therelevant hybridization assay. For example, those in which the stringencyof hybridization and wash conditions are increased until the signal tonoise ratio for binding of the probe to the perfectly matchedcomplementary target nucleic acid is at least 10×, 20×, 50×, 100×, or500× or more as high as that observed for hybridization to any of theunmatched target nucleic acids. A target nucleic acid which hybridizesto a probe under such conditions, with a signal to noise ratio of atleast, that of the perfectly matched complementary target nucleic acidis said to bind to the probe under ultra-ultra-high stringencyconditions.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, e.g., when a copyof a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code.

Unique Subsequences

In one aspect, the invention provides a nucleic acid that comprises aunique subsequence in a nucleic acid selected from the sequences ofO-tRNA's and O-RSs disclosed herein. The unique subsequence is unique ascompared to a nucleic acid corresponding to any known O-tRNA or O-RSnucleic acid sequence. Alignment can be performed using, e.g., BLAST setto default parameters. Any unique subsequence is useful, e.g., as aprobe to identify the nucleic acids of the invention.

Similarly, the invention includes a polypeptide which comprises a uniquesubsequence in a polypeptide selected from the sequences of O-RSsdisclosed herein. Here, the unique subsequence is unique as compared toa polypeptide corresponding to any known polypeptide sequence.

The invention also provides for target nucleic acids which hybridizesunder stringent conditions to a unique coding oligonucleotide whichencodes a unique subsequence in a polypeptide selected from thesequences of O-RSs wherein the unique subsequence is unique as comparedto a polypeptide corresponding to any of the control polypeptides (e.g.,parental sequences from which synthetases of the invention were derived,e.g., by mutation). Unique sequences are determined as noted above.

Sequence Comparison, Identity, and Homology

The terms “identical” or percent “identity,” in the context of two ormore nucleic acid or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the sequence comparison algorithms described below (or otheralgorithms available to persons of skill) or by visual inspection.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides (e.g., DNAs encoding an O-tRNA or O-RS, or theamino acid sequence of an O-RS) refers to two or more sequences orsubsequences that have at least about 60%, preferably 80%, mostpreferably 90-95% nucleotide or amino acid residue identity, whencompared and aligned for maximum correspondence, as measured using asequence comparison algorithm or by visual inspection. Such“substantially identical” sequences are typically considered to be“homologous,” without reference to actual ancestry. Preferably, the“substantial identity” exists over a region of the sequences that is atleast about 50 residues in length, more preferably over a region of atleast about 100 residues, and most preferably, the sequences aresubstantially identical over at least about 150 residues, or over thefull length of the two sequences to be compared.

For sequence comparison and homology determination, typically onesequence acts as a reference sequence to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are input into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. The sequence comparison algorithm then calculates thepercent sequence identity for the test sequence(s) relative to thereference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generally,Ausubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are then extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M-5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

Mutagenesis and Other Molecular Biology Techniques

General texts which describe molecular biological techniques includeBerger and Kimmel, Guide to Molecular Cloning Techniques. Methods inEnzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger);Sambrook et al., Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989(“Sambrook”) and Current Protocols in Molecular Biology, F. M. Ausubelet al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (supplementedthrough 1999) (“Ausubel”)). These texts describe mutagenesis, the use ofvectors, promoters and many other relevant topics related to, e.g., thegeneration of genes that include selector codons for production ofproteins that include unnatural amino acids, orthogonal tRNA's,orthogonal synthetases, and pairs thereof.

Various types of mutagenesis are used in the invention, e.g., to producelibraries of tRNA's, to produce libraries of synthetases, to insertselector codons that encode unnatural amino acids in a protein orpolypeptide of interest. They include but are not limited tosite-directed, random point mutagenesis, homologous recombination, DNAshuffling or other recursive mutagenesis methods, chimeric construction,mutagenesis using uracil containing templates, oligonucleotide-directedmutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesisusing gapped duplex DNA or the like, or any combination thereof.Additional suitable methods include point mismatch repair, mutagenesisusing repair-deficient host strains, restriction-selection andrestriction-purification, deletion mutagenesis, mutagenesis by totalgene synthesis, double-strand break repair, and the like. Mutagenesis,e.g., involving chimeric constructs, are also included in the presentinvention. In one embodiment, mutagenesis can be guided by knowninformation of the naturally occurring molecule or altered or mutatednaturally occurring molecule, e.g., sequence, sequence comparisons,physical properties, crystal structure or the like.

The above texts and examples found herein describe these procedures.Additional information is found in the following publications andreferences cited within: Ling at al., Approaches to DNA mutagenesis: anoverview, Anal Biochem. 254(2): 157-178 (1997); Dale et al.,Oligonucleotide-directed random mutagenesis using the phosphorothioatemethod, Methods Mol. Biol. 57:369-374 (1996); Smith, In vitromutagenesis, Ann. Rev. Genet. 19:423-462(1985); Botstein & Shortle,Strategies and applications of in vitro mutagenesis, Science229:1193-1201(1985); Carter, Site-directed mutagenesis, Biochem. J.237:1-7 (1986); Kunkel, The efficiency of oligonucleotide directedmutagenesis, in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin)) (1987); Kunkel, Rapidand efficient site-specific mutagenesis without phenotypic selection,Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel at al., Rapid andefficient site-specific mutagenesis without phenotypic selection,Methods in Enzymol. 154, 367-382 (1987); Bass at al., Mutant Trprepressors with new DNA-binding specificities, Science 242:240-245(1988); Methods in Enzymol. 100: 468-500 (1983); Methods in Enzymol.154: 329-350 (1987); Zoller & Smith, Oligonucleotide-directedmutagenesis using M13-derived vectors: an efficient and generalprocedure for the production of point mutations in any DNA fragment,Nucleic Acids Res. 10:6487-6500 (1982); Zoller & Smith,Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors, Methods in Enzymol. 100:468-500 (1983); Zoller & Smith,Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template, Methods inEnzymol. 154:329-350 (1987); Taylor at al., The use ofphosphorothioate-modified DNA in restriction enzyme reactions to preparenicked DNA, Nucl. Acids Res. 13: 8749-8764 (1985); Taylor et al., Therapid generation of oligonucleotide-directed mutations at high frequencyusing phosphorothioate-modified DNA, Nucl. Acids Res. 13: 8765-8787(1985); Nakamaye & Eckstein, Inhibition of restriction endonuclease NciI cleavage by phosphorothioate groups and its application tooligonucleotide-directed mutagenesis, Nucl. Acids Res. 14: 9679-9698(1986); Sayers et al., Y-T Exonucleases in phosphorothioate-basedoligonucleotide-directed mutagenesis, Nucl. Acids Res. 16:791-802(1988); Sayers et al., Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide, (1988) Nucl. AcidsRes. 16: 803-814; Kramer at al., The gapped duplex DNA approach tooligonucleotide-directed mutation construction, Nucl. Acids Res. 12:9441-9456 (1984); Kramer & Fritz Oligonucleotide-directed constructionof mutations via gapped duplex DNA, Methods in Enzymol. 154:350-367(1987); Kramer at al., Improved enzymatic in vitro reactions in thegapped duplex DNA approach to oligonucleotide-directed construction ofmutations, Nucl. Acids Res. 16: 7207 (1988); Fritz et al.,Oligonucleotide-directed construction of mutations: a gapped duplex DNAprocedure without enzymatic reactions in vitro, Nucl. Acids Res. 16:6987-6999 (1988); Kramer at al., Point Mismatch Repair, Cell 38:879-887(1984); Carter et al., Improved oligonucleotide site-directedmutagenesis using M13 vectors, Nucl. Acids Res. 13: 4431-4443 (1985);Carter, Improved oligonucleotide-directed mutagenesis using M13 vectors,Methods in Enzymol. 154: 382-403 (1987); Eghtedarzadeh & Henikoff, Useof oligonucleotides to generate large deletions, Nucl. Acids Res. 14:5115 (1986); Wells et al., Importance of hydrogen-bond formation instabilizing the transition state of subtilisin, Phil. Trans. R. Soc.Lond. A 317: 415-423 (1986); Nambiar et al., Total synthesis and cloningof a gene coding for the ribonuclease S protein, Science 223: 1299-1301(1984); Sakamar and Khorana, Total synthesis and expression of a genefor the a-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin), Nucl. Acids Res. 14: 6361-6372 (1988); Wells etal., Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites, Gene 34:315-323 (1985); Grundströmet al., Oligonucleotide-directed mutagenesis by microscale ‘shot-gun’gene synthesis, Nucl. Acids Res. 13: 3305-3316 (1985); Mandecki,Oligonucleotide-directed double-strand break repair in plasmids ofEscherichia coli: a method for site-specific mutagenesis, Proc. Natl.Acad. Sci. USA, 83:7177-7181 (1986); Arnold, Protein engineering forunusual environments, Current Opinion in Biotechnology 4:450-455 (1993);Sieber, et al., Nature Biotechnology, 19:456-460 (2001). W. P. C.Stemmer, Nature 370, 389-91 (1994); and, I. A. Lorimer, I. Pastan,Nucleic Acids Res. 23, 3067-8 (1995). Additional details on many of theabove methods can be found in Methods in Enzymology Volume 154, whichalso describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

The invention also relates to vertebrate host cells and organisms forthe in vivo incorporation of an unnatural amino acid via orthogonaltRNA/RS pairs. Host cells are genetically engineered (e.g., transformed,transduced or transfected) with the polynucleotides of the invention orconstructs which include a polynucleotide of the invention, e.g., avector of the invention, which can be, for example, a cloning vector oran expression vector. The vector can be, for example, in the form of aplasmid, a bacterium, a virus, a naked polynucleotide, or a conjugatedpolynucleotide. The vectors are introduced into cells and/ormicroorganisms by standard methods including electroporation (From etal., Proc. Natl. Acad. Sci. USA 82, 5824 (1985), infection by viralvectors, high velocity ballistic penetration by small particles with thenucleic acid either within the matrix of small beads or particles, or onthe surface (Klein et al., Nature 327, 70-73 (1987)).

The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for such activities as, for example, screeningsteps, activating promoters or selecting transformants. These cells canoptionally be cultured into transgenic organisms. Other usefulreferences, e.g. for cell isolation and culture (e.g., for subsequentnucleic acid isolation) include Freshney (1994) Culture of Animal Cells.a Manual of Basic Technique, third edition, Wiley-Liss, New York and thereferences cited therein; Payne et al. (1992) Plant Cell and TissueCulture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.;Gamborg and Phillips (eds) (1995) Plant Cell Tissue and Organ Culture;Fundamental Methods Springer Lab Manual, Springer-Verlag (BerlinHeidelberg New York) and Atlas and Parks (eds) The Handbook ofMicrobiological Media (1993) CRC Press, Boca Raton, Fla.

The invention also relates to vertebrate cell lines with the ability toincorporate an unnatural amino acid or acids via orthogonal tRNA/RSpairs. These cell lines can be established using cell culture techniquesknown in the art on host cells which have been transformed, transduced,or transfected with the polynucleotides of the invention or constructswhich include a polynucleotide of the invention. The methods ofintroducing exogenous nucleic acids into host cells are well known inthe art, and will vary with the host cell used. Techniques include, butare not limited to, dextran-mediated transfection, calcium phosphateprecipitation, calcium chloride treatment, polybrene mediatedtransfection, protoplast fusion, electroporation, viral or phageinfection, encapsulation of the polynucleotide(s) in liposomes, anddirect microinjection.

Cells may be transformed or transfected in a manner to allow eithertransient or stable incorporation of DNA. For long-term, high-yieldproduction of recombinant proteins, stable expression is preferred. Forexample, cell lines which stably express the antibody molecule may beengineered. Rather than using expression vectors which contain viralorigins of replication, host cells can be transformed with DNAcontrolled by appropriate expression control elements (e.g., promoter,enhancer, sequences, transcription terminators, polyadenylation sites,etc.), and a selectable marker. Following the introduction of theforeign DNA, engineered cells may be allowed to grow for 1-2 days in anenriched media, and then are switched to a selective media. Theselectable marker in the recombinant plasmid confers resistance to theselection and allows cells to stably integrate the plasmid into theirchromosomes and grow to form foci which in turn can be cloned andexpanded into cell lines. This method may advantageously be used toengineer cell lines which express the antibody molecule. Such engineeredcell lines may be particularly useful in screening and evaluation ofcompounds that interact directly or indirectly with the antibodymolecule. Alternatively, other techniques, such as some viral-mediatedvector transfection techniques, well known to those in the art, canpermit transient transfection of cells.

Several well-known methods of introducing target nucleic acids intocells are available, any of which can be used in the invention. Theseinclude: fusion of the recipient cells with bacterial protoplastscontaining the DNA, electroporation, projectile bombardment (for morestable expression), and infection with viral vectors (which can be usedfor stable or transient transfection and which is also discussedfurther, below), etc. Bacterial cells can be used to amplify the numberof plasmids containing DNA constructs of this invention. The bacteriaare grown to log phase and the plasmids within the bacteria can beisolated by a variety of methods known in the art (see, for instance,Sambrook). In addition, a plethora of kits are commercially availablefor the purification of plasmids from bacteria, (see, e.g., EasyPrep™,FlexiPrep™, both from Pharmacia Biotech; StrataClean™, from Stratagene;and, QIAprep™ from Qiagen). The isolated and purified plasmids are thenfurther manipulated to produce other plasmids, used to transfect cellsor incorporated into related vectors to infect organisms. Typicalvectors contain transcription and translation terminators, transcriptionand translation initiation sequences, and promoters useful forregulation of the expression of the particular target nucleic acid. Thevectors optionally comprise generic expression cassettes containing atleast one independent terminator sequence, sequences permittingreplication of the cassette in eukaryotes, or prokaryotes, or both,(e.g., shuttle vectors) and selection markers for both prokaryotic andvertebrate systems. Vectors are suitable for replication and integrationin prokaryotes, eukaryotes, or preferably both. See, Giliman & Smith,Gene 8:81 (1979); Roberts, et al., Nature, 328:731 (1987); Schneider,B., et al., Protein Expr. Purif. 6435:10 (1995); Ausubel, Sambrook,Berger (all supra). A catalogue of Bacteria and Bacteriophages usefulfor cloning is provided, e.g., by the ATCC, e.g., The ATCC Catalogue ofBacteria and Bacteriohage (1992) Gherna et al. (eds) published by theATCC. Additional basic procedures for sequencing, cloning and otheraspects of molecular biology and underlying theoretical considerationsare also found in Watson et al. (1992) Recombinant DNA Second EditionScientific American Books, NY. In addition, essentially any nucleic acid(and virtually any labeled nucleic acid, whether standard ornon-standard) can be custom or standard ordered from any of a variety ofcommercial sources, such as the Midland Certified Reagent Company(Midland, Tex. mcrc.com), The Great American Gene Company (Ramona,Calif. available on the World Wide Web at genco.com), ExpressGen Inc.(Chicago, Ill. available on the World Wide Web at expressgen.com),Operon Technologies Inc. (Alameda, Calif.) and many others.

Kits

Kits are also a feature of the invention. For example, a kit forproducing a protein that comprises at least one unnatural amino acid ina cell is provided, where the kit includes a container containing apolynucleotide sequence encoding an O-tRNA, and/or an O-tRNA, and/or apolynucleotide sequence encoding an O-RS, and/or an O-RS. In oneembodiment, the kit further includes at least one unnatural amino acid.In another embodiment, the kit further comprises instructional materialsfor producing the protein.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention. One of skill will recognize a variety of non-criticalparameters that may be altered without departing from the scope of theclaimed invention.

Example 1

Methods of Producing and Compositions of Aminoacyl-tRNA Synthetases thatIncorporate Unnatural Amino Acids in Vertebrate Cells

The expansion of the vertebrate genetic code to include unnatural aminoacids with novel physical, chemical or biological properties wouldprovide powerful tools for analyzing and controlling protein function inthese cells. Towards this goal, a general approach for the isolation ofaminoacyl-tRNA synthetases that incorporate unnatural amino acids withhigh fidelity into proteins in response to an amber codon inSaccharomyces cerevisiae (S. cerevisiae) is described. The method isbased on the activation of GAL4 responsive reporter genes, HIS3, URA3 orLacZ, by suppression of amber codons between the DNA binding domain andtranscriptional activation domain of GAL4. The optimization of a GAL4reporter for positive selection of active Escherichia coli tyrosyl-tRNAsynthetase (EcTyrRS) variants is described. A negative selection ofinactive EcTyrRS variants has also been developed with the URA3 reporterby use of a small molecule (5-fluroorotic acid (5-FOA)) added to thegrowth media as a ‘toxic allele.’ Importantly both positive and negativeselections can be performed on a single cell and with a range ofstringencies. This can facilitate the isolation of a range ofaminoacyl-tRNA synthetase (aaRS) activities from large libraries ofmutant synthetases. The power of the method for isolating desired aaRSphenotypes is demonstrated by model selections.

Example 2

E. coli and B. stearothermophilus Tyr tRNA Hybrid tRNA Construction

It is known from work in Saccharomyces cerevisiae, that the E. coli TyrtRNA/RS pairs are orthogonal to endogenous tRNA/RS pairs and supportsunnatural amino acid suppression. However, efforts to transcribefunctional E. coli tRNA^(Tyr) in vivo in mammalian cells have beenchallenging. Because of this, interest has turned to B.stearothermophilus as a source of tRNA sequence that can supportunnatural amino acid suppression in mammalian cells. Though B.stearothermophilus tRNA is a substrate for E. coli tRNA^(Tyr)synthetase, further engineering of tRNA is needed to improve the tRNAaminoacylation efficiency. Improved tRNA aminoacylation will improvesuppression efficiency. The acceptor stem of the tRNA is a keydeterminant for tRNA synthetase recognition. In this example, a hybridtRNA was constructed by combining different structural components of E.coli and B. stearothermophilus tRNA^(Tyr). This hybrid tRNA has theacceptor stem of E. coli tRNA^(Tyr), and the D arm, TψC arm, variableloop and anticodon stem of B. stearothermophilus tRNA^(Tyr). The newhybrid tRNA, having an acceptor stem that derives from E. coli, is abetter substrate for E. coli tRNA^(Tyr) synthetase. We show in theexperiment below that improved amber suppression efficiency was obtainedwhen the newly created hybrid amber-suppressing tRNA was used. Forcomparison, the hybrid tRNA was tested alongside the B.stearothermophilus tRNA^(Tyr) from which it was derived.

Experimental:

Construction of Plasmid Encoding Hybrid tRNA:

Single-copy hybrid amber-suppressing tRNA expression insert whichincludes 5′ restriction sites (EcoR I and Bgl II), 5′ flanking sequenceof human tRNA^(Tyr)(GGATACGCATGCTCAGTGCAATCTrCGGTTGCCTGGACTAOCGCTCCGGTTTTTCTOTGCTGAACCTCAGGOGACOCCGACACACOTACACGTC (SEQ ID NO: 89)), the hybridtRNA amber suppression mutant lacking 3′-CCA (The nucleotide sequence ofthe hybrid tRNA is as follows:GOUGGGGUAGCGAAGUGGCUAAACGCGGCGGACUCUAAAUCCOCUCCCUUUGGGUUCGGCGGTUCGAAUCCGUCCCCCUCCACCA (SEQ ID NO:87), and the DNA sequenceencoding the tRNA is as follows:GGTGGGTAGCGAAGTGGCTAAACGCGGCGGACTCTAAATCCGCTCCCTTGGGTTCGGCGGTTCGAATCCGTCCCCCA (SEQ ID NO: 88)), 3′ flanking sequence ofhuman tRNA^(Tyr) (GACAAGTGCGGTTTTTTTCTCCAGCTCCCGATGACTTATGGC (SEQ ID NO:90)) and 3′ restriction sites (BamH I and Hind III), was constructed byoverlap PCR using primers:

FTam 73: forward primer with EcoR I and Bgl II site (SEQ ID NO: 91)GTACGAATTCCCGAGATCTGGATTACGCATGCTCAGTGCAATCTTCGGTTGCCTGGACTAGCGCTCCGGTTTTTCTGTGC FTam 115: reverse primer: (SEQ ID NO: 92)AGTCCGCCGCGTTTAGCCACTTCGCTACCCCACCGACGTGTACGTGTGTCGGCGTCCCCTGAGGTTCAGCACAGAAAAACCGGAGCGCFTam116: forward primer for picec 2: (SEQ ID NO: 93)GTGGCTAAACGCGGCGGACTCTAAATCCGCTCCCTTTGGGTTCGGCGGTTCGAATCCGTCCCCCACCAGACAAGTG FTam117: reverse primer for piece 2:(SEQ ID NO: 94) GATGCAAGCTTGATGGATCCGCCATAAGTCATCGGGAGCTGGAGAAAAAAACCGCACTTGTCTGGTGGGGGACGG.The insert was ligated into pUC19 at EcoR I and Hind III sites.

Amber suppression experiment with hybrid tRNA (FIG. 1):

Plasmids encoding the hGH E88 amber mutant, E. coli tRNA synthetase andeither the single-copy amber-suppressing B. stearothermophilus tRNA orthe single-copy amber-suppressing hybrid tRNA were co-transfected intoCHO K1 cells. The expression of hGH was assayed 42 hours aftertransfection. When the hybrid tRNA (hb1) was used, amber suppressionefficiency increased approximately 30% relative to that obtained whenthe B. stearothermophilus amber suppressing tRNA was used.

Example 3

Addition of molecules to proteins with an unnatural amino acid.

In one aspect, the invention provides methods and related compositionsof proteins comprising unnatural amino acids coupled to additionalsubstituent molecules.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus describedherein can be used in various combinations. All publications, patents,patent applications, and/or other documents cited in this applicationare incorporated by reference in their entirety for all purposes to thesame extent as if each individual publication, patent, patentapplication, and/or other document were individually indicated to beincorporated by reference for all purposes.

TABLE 5  SEQ ID NO.: Label SEQUENCE SEQ ID E. coli wild-ATGGCAAGCAGTAACTTGATTAAACAATTGCAAGAGCGGGGGCTGGTA NO.: 1 type TyrRSGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGG (synthetase)CCCGATCGCGCTCTATTGCGGCTTCGATCCTACCGCTGACAGCTTGCAT poly-TTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGG nucleotideGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCGAACAACTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGTTGCAGGGTTATGACTTCGCCTGTCTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTGTTTGGCCTGACCGTTCCGCTGATCACTAAAGCAGATGGCACCAAATTTGGTAAAACTGAAGGCGGCGCAGTCTGGTTGGATCCGAAGAAAACCAGCCCGTACAAATTCTACCAGTTCTGGATCAACACTGCGGATGCCGACGTTTACCGCTTCCTGAAGTTCTTCACCTTTATGAGCATTGAAGAGATCAACGCCCTGGAAGAAGAAGATAAAAACAGCGGTAAAGCACCGCGCGCCCAGTATGTACTGGCGGAGCAGGTGACTCGTCTGGTTCACCGTGAAGAAGGTTTACAGGCGGCAAAACGTATTACCGAATGCCTGTTCAGCGGTTCTTTGAGTGCGCTGAGTGAAGCGGACTTCGAACAGCTGGCGCAGGACGGCGTACCGATGGTTGAGATGGAAAAGGGCGCAGACCTGATGCAGGCACTGGTCGATTCTGAACTGCAACCTTCCCGTGGTCAGGCACGTAAAACTATCGCCTCCAATGCCATCACCATTAACGGTGAAAAACAGTCCGATCCTGAATACTTCTTTAAAGAAGAAGATCGTCTGTTTGGTCGTTTTACCTTACTGCGTCGCGGTAAAAAGAATTACTGT CTGATTTGCTGGAAATAASEQ ID E. coli wild- MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALYCGFDPTADSLHLGHNO.: 2 type TyrRS LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWV(synthetase) DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMAmino acid INKEAVKQRLNREDQGISFTEFSYNLLQGYDFACLNKQYGVVLQIGGSDQ (aa)WGNITSGIDLIRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID pOMe-1 ATGGCAAGCAGTAACTTGATTAAACAATTGCAAGAGCGGGGGCTGGTA NO.: 3Synthetase GCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGGC poly-CCGATCGCACTCGTGTGTGGCTTCGATCCTACCGCTGACAGCTTGCATTT nucleotideGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATAGTATGGCCTGTTTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTGTTTGGCCTGACCGTTCCGCTGATCACTAAAGCAGATGGCACCAAATTTGGTAAAACTGAAGGCGGCGCAGTCTGGTTGGATCCGAAGAAAACCAGCCCGTACAAATTCTACCAGTTCTGGATCAACACTGCGGATGCCGACGTTTACCGCTTCCTGAAGTTCTTCACCTTTATGAGCATTGAAGAGATCAACGCCCTGGAAGAAGAAGATAAAAACAGCGGTAAAGCACCGCGCGCCCAGTATGTACTGGCGGAGCAGGTGACTCGTCTGGTTCACGGTGAAGAAGGTTTACAGGCGGCAAAACGTATTACCGAATGCCTGTTCAGCGGTTCTTTGAGTGCGCTGAGTGAAGCGGACTTCGAACAGCTGGCGCAGGACGGCGTACCGATGGTTGAGATGGAAAAGGGCGCAGACCTGATGCAGGCACTGGTCGATTCTGAACTGCAACCTTCCeGTGGTCAGGCACGTAAAACTATCGCCTCCAATGCCATCACCATTAACGGTGAAAAACAGTCCGATCCTGAATACTTCTTTAAAGAAGAAGATCGTCTGTTTGGTCGTTTTACCTTACTGCGTCGCGGTAAAAAGAATTACTGTCTGATTTGC TGGAAATAA SEQ IDpOMe-2 ATGGCAAGCAGTAACTTGATTAAACAATTGCAAGAGCGGGGGCTGGTA NO.: 4Synthetase gCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGGC poly-CCGATCGCACTCACTTGTGGCTTCGATCCTACCGCTGACAGCTTGCATTT nucleotideGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCAGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATACGTATGCCTGTCTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTGTTTGGCCTGACCGTTCCGCTGATCACTAAAGCAGATGGCACCAAATTTGGTAAAACTGAAGGCGGCGCAGTCTGGTTGGATCCGAAGAAAACCAGCCCGTACAAATTCTACCAGTTCTGGATCAACACTGCGGATGCCGACGTTTACCGCTTCCTGAAGTTCTTCACCTTTATGAGCATTGAAGAGATCAACGCCCTGGAAGAAGAAGATAAAAACAGCGGTAAAGCACCGCGCGCCCAGTATGTACTGGCGGAGCAGGTGACTCGTCTGGTTCACGGTGAAGAAGGTTTACAGGCGGCAAAACGTATTACCGAATGCCTGTTCAGCGGTTCTTTGAGTGCGCTGAGTGAAGCGGACTTCGAACAGCTGGCGCAGGACGGCGTACCGATGGTTGAGATGGAAAAGGGCGCAGACCTGATGCAGGCACTGGTCGATTCTGAACTGCAACCTTCCCGTGGTCAGGCACGTAAAACTATCGCCTCCAATGCCATCACCATTAACGGTGAAAAACAGTCCGATCCTGAATACTTCTTTAAAGAAGAAGATCGTCTGTTTGGTCGTTTTACCTTACTGCGTCGCGGTAAAAAGAATTACTGTCTGATTTGC TGGAAATAA SEQ IDpOMe-3 ATGGCAAGCAGTAACTTGATTAAACAATTGCAAGAGCGGGGGCTGGTA NO.: 5Synthetase GCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGGC poly-CCGATCGCACTCGTGTGTGGCTTCGATCCTACCGCTGACAGCTTGCATTT nucleotideGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATAGTATGGCCTGTTTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTGTTTGGCCTGACCGTTCCGCTGATCACTAAAGCAGATGGCACCAAATTTGGTAAAACTGAAGGCGGCGCAGTCTGGTTGGATCCGAAGAAAACCAGCCCGTACAAATTCTACCAGTTCTGGATCAACACTGCGGATGCCGACGTTTACCGCTTCCTGAAGTTCTTCACCTTTATGAGCATTGAAGAGATCAACGCCCTGGAAGAAGAAGATAAAAACAGCGGTAAAGCACCGCGCGCCCAGTATGTACTGGCGGAGCAGGTGACTCGTCTGGTTCACGGTGAAGAAGGTTTACAGGCGGCAAAACGTATTACCGAATGCCTGTTCAGCGGTTCTTTGAGTGCGCTGAGTGAAGCGGACTTCGAACAGCTGGCGCAGGACGGCGTACCGATGGTTGAGATGGAAAAGGGCGCAGACCTGATGCAGGCACTGGTCGATTCTGAACTGCAACCTTCCCGTGGTCAGGCACGTAAAACTATCGCCTCCAATGCCATCACCATTAACGGTGAAAAACAGTCCGATCCTGAATACTTCTTTAAAGAAGAAGATCGTCTGTTTGGTCGTTTTACCTTACTGCGTCGCGGTAAAAAGAATTACTGTCTGATTTGC TGGAAATAA SEQ IDpOMe-4 ATGGCAAGCAGTAACTTGATTAAACAATTGCAAGAgCGGGGGCTGGTA NO.: 6Synthetase GCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGGC poly-CCGATCGCACTCGTGTGTGGCTTCGATCCTACCGCTGACAGCTTGCATTT nucleotideGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATAGTATGGCCTGTTTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTGTTTGGCCTGACCGTTCCGCTGATCACTAAAGCAGATGGCACCAAATTTGGTAAAACTGAAGGCGGCGCAGTCTGGTTGGATCCGAAGAAAACCAGCCCGTACAAATTCTACCAGTTCTGGATCAACACTGCGGATGCCGACGTTTACCGCTTCCTGAAGTTCTTCACCTTTATGAGCATTGAAGAGATCAACGCCCTGGAAGAAGAAGATAAAAACAGCGGTAAAGCACCGCGCGCCCAGTATGTACTGGCGGAGCAGGTGACTCGTCTGGTTCACCGTGAAGAAGGTTTACAGGCGGCAAAACGTATTACCGAATGCCTGTTCAGCGGTTCTTTGAGTGCGCTGAGTGAAgCGGACTTCGAACAGCTGGCGCAGGACGGCGTACCGATGGTTGAGATGGAAAAGGGCGCAGACCTGATGCAGGCACTGGTCGATTCTGAACTGCAACCTTCCCGTGGTCAGGCACGTAAAACTATCGCCTCCAATGCCATCACCATTAACGGTGAAAAACAGTCCGATCCTGAATACTTCTTTAAAGAAGAAGATCGTCTGTTTGGTCGTTTTACCTTACTGCGTCGCGGTAAAAAGAATTACTGTCTGATTTGC TGGAAATAA SEQ IDpOMe-5 ATGGCAAGCAGTAACTTGATTAAACAATTGCAAGAGCGGGGGCTGGTA NO.: 7Synthetase gCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGGC poly-CCGATCGCACTCACGTGTGGCTTCGATCCTACCGCTGACAGCTTGCATT nucleotideTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAGCCTGCTGCAGGGTTATACGATGGCCTGTCTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTGTTTGGCCTGACCGTTCCGCTGATCACTAAAGCAGATGGCACCAAATTTGGTAAAACTGAAGGCGGCGCAGTCTGGTTGGATCCGAAGAAAACCAGCCCGTACAAATTCTACCAGTTCTGGATCAACACTGCGGATGCCGACGTTTACCGCTTCCTGAAGTTCTTCACCTTTATGAGCATTGAAGAGATCAACGCCCTGGAAGAAGAAGATAAAAACAGCGGTAAAGCACCGCGCGCCCAGTATGTACTGGCGGAGCAGGTGACTCGTCTGGTTCACGGTGAAGAAGGTTTACAGGCGGCAAAACGTATTACCGAATGCCTGTTCAGCGGTTCTTTGAGTGCGCTGAGTGAAGCGGACTTCGAACAGCTGGCGCAGGACGGCGTACCGATGGTTGAGATGGAAAAGGGCGCAGACCTGATGCAGGCACTGGTCGATTCTGAACTGCAACCTTCCCGTGGTCAGGCACGTAAAACTATCGCCTCCAATGCCATCACCATTAACGGTGAAAAACAGTCCGATCCTGAATACTTCTTTAAAGAAGAAGATCGTCTGTTTGGTCGTTTTACCTTACTGCGTCGCGGTAAAAAGAATTACTGTCTGATTTGC TGGAAATAA SEQ IDpOMe-6 CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 8(active site) CTGGCGCAAGGCCCGATCGCACTCACTTGTGGCTTCGATCCTACCGCTGSynthetase ACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC poly-CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT nucleotideCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCAGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATACGTATGCCTGTCTGAACAAACAGTACG GTGTG SEQ ID pOMe-7CGGGGGCTGGTACCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 9 (active site)CTGGCGCAAGGCCCGATCGCACTCACTTGTGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC poly-CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT nucleotideCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCAGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATACGTATGCCTGTCTGAACAAACAGTACG GTGTG SEQ ID pOMe-8CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 10 (active site)CTGGCGCAAGGCCCGATCGCACTCACTTGTGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC poly-CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT nucleotideCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCAGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATACGTATGCCTGTCTGAACAAACAGTACG GTGTG SEQ ID pOMe-9CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 11 (active site)CTGGCGCAAGGCCCGATCGCACTCACTTGTGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC poly-CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT nucleotideCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATTCGTATGCCTGTGCGAACAAACAGTACG GTGTG SEQ ID pOMe-10CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 12 (active site)CTGGCGCAAGGCCCGATCGCACTCACTTGTGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC poly-CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT nucleotideCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCAGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATACGTATGCCTGTCTGAACAAACAGTACG GTGTG SEQ ID pOMe-11CGGGGGCTGGTACCcCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 13 (active site)CTGGCGCAAGGCCCGATCGCACTCCTTTGTGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC poly-CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT nucleotideCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTMCCTACAACCTGCTGCAGGGTTATTCTATTGCCTGTTCGAACAAACAGTACG GTGTG SEQ ID pOMe-12CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 14 (active site)CTGGCGCAAGGCCCGATCGCACTCGTGTGTGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC poly-CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT nucleotideCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTMCCTACAACCTGCTGCAGGGTTATAGTATTGCCTGTTTGAACAAACAGTACG GTGTG SEQ ID pOMe-13CGGGGGCTGGTACCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 15 (active site)CTGGCGCAAGGCCCGATCGCACTCGTGTGTGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC poly-CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT nucleotideCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATAGTATTGCCTGTTTGAACAAACAGTACG GTGTG SEQ ID pOMe-14CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 16 (active site)CTGGCGCAAGGCCCGATCGCACTCTGGTGTGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC poly-CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT nucleotideCTGATTGGCGACCCGAGCTTCAAGGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATTGTTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATATGCGTGCCTGTGAGAACAAACAGTACG GTGTG SEQ IDp-acetylPhe-1 CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 17(active site) CTGGCGCAAGGCCCGATCGCACTCATTTGTGGCTTCGATCCTACCGCTGSynthetase ACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC poly-CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT nucleotideCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGGTCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATGGTATGGCCTGTGCTAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAATGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTG SEQ ID pBenzophenon-CAGGTGACGGACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGGCCCG NO.: 18 1 (active ATCGCACTCGGTTGTGGCTTCGATCCTACCGCTGACAGCTTGCATTTGG site) Syn-GGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGCCA thetaseCAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCC polynucleotideGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATGGTTTTGCCTGTTTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTG SEQ ID pBenzophenone-GCGTTAGCAGAGCGACTGGCGCAAGGCCCGATCGCACTCGGGTGTGGC NO.: 19 2 (active TTCGATCCTACCGCTGACAGCTTGCATTTGGGGCATCTTGTTCCATTGTT site) Syn-ATGCCTGAAACGCTTCCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGT thetaseAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGA polynucleotideGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATGGTTATGCCTGTATGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAAT CAGGTG SEQ IDpAzidoPhe-1 GGGCTGGTAGCCCAGGTGACGGACGNAGAAGCGTTAGCAGAGCGACTG NO.: 20(active site) GCGCAAGGCCCGATCGCACTCCTTTGTGGCTTCGATCCTACCGCTGACASynthetase GCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCCAG poly-CAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTG nucleotideATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATTCTATGGCCTGTGCGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCANAATCANGTG SEQ ID pAzidoPhe-2TTAGCAGAGCGACTGGCGCAAGGCCCGATCGCACTCGTTTGTGGCTTCG NO.: 21 (active site)ATCCTACCGCTGACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGC SynthetaseCTGAAACGCTTCCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGC poly-GGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGT nucleotideAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATTCTGCGGCCTGTGCGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCA GGTG SEQ IDpAzidoPhe-3 GACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGGCCCGATCGCACTC NO.: 22(active site) CTGTGTGGCTTCGATCCTACCGCTGACAGCTTGCATTTGGGGCATCTTGTSynthetase TCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGCCACAAGCCGGTT poly-GCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAA nucleotideGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAANAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATTCGGCTGCCTGTGCGAACAAACAGTACGGNGNGGNGCTGCAAATTGGNGGTTCTGACCAGGGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTG CATCAAAATCAGGTG SEQ IDpAzidoPhe-4 GCGTTAGCAGAGCGACTGGCGCAAGGCCCGATCGCACTCGTTTGTGGCT NO.: 23(active site) TCGATCCTACCGCTGACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTGSynthetase TGCCTGAAACGCTTCCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTA poly-GGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAG nucleotideCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATAGTGCGGCCTGTGTTAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACGAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATC ANGTG SEQ IDpAzidoPhe-5  GACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGGCCCGATCGCACTC NO.: 24(active site) ATTTGTGGCTTCGATCCTACCGCTGACAGCTTGCATTTGGGGCATCTTGTSynthetase TCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGCCACAAGCCGGTT poly-GCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAA nucleotideGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATGATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATAATTTTGCCTGTGTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGC ATCAGAATCAGGTG SEQ IDpAzidoPhe-6 CGACTGGCGCAAGGCCCGATCGCACTCACGTGTGGCTTCGATCCTACCG NO.: 25(active site) CTGACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCSynthetase TTCCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACG poly-GGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAAC nucleotideACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAATCTGCTGCAGGGTTATTCGGCTGCCTGTCTTAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTG SEQ ID pPR-EcRS-1CGGGGGCTGGTANCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 26 (propargyloxyCTGGCGCAAGGCCCGATCGCACTCGGGTGTGGCTTCGATCCTACCGCTG phenylalanineACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC synthetase) CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT (active site)CTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACC SynthetaseGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCC poly-CCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATA nucleotideATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATTCTATGGCCTGTTTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGANCCGTCGTCTGCATCAGAATCAGGTG SEQ ID pPR-EcRS-2CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 27 (active site)CTGGCGCAAGGCCCGATCGCACTCACGTGTGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC poly-CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT nucleotideCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAATCTGCTGCAGGGTTATTCGGCTGCCTGTCTTAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGAACCTGANCCGTCGTCTGCATCAAAATCAAGTG SEQ ID pPR-EcRS-3CGGGGGCTGGTACCCCAAGTGACGGACGAGGAAACGTTAGCAGAGCGA NO.: 28 (active site)CTGGCGCAAGGCCCGATCGCACTCTCTTGTGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC poly-CAGCAGGCAGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT nucleotideCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATACGATGGCCTGTGTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTG SEQ ID pPR-EcRS-4CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 29 (active site)CTGGCGCAAGGCCCGATCGCACTCGCGTGCGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC poly-CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT nucleotideCTGATTGGCGACCCGAGCTTCAAGGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATTCTTATGCCTGTCTTAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTG SEQ ID pPR-EcRS-5CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 30 (active site)CTGGCGCAAGGCCCGATCGCACTCGCGTGTGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC poly-CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT nucleotideCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATACGATGGCCTGTTGTAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTG SEQ ID pPR-EcRS-6CGGGGGCTGGTACCCCAAGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 31 (active site)CTGGCGCAAGGCCCGATCGCACTCACGTGTGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC poly-CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT nucleotideCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCGCTGAGTTTTCCTACAACCTGCTGCAGGGTTATACGTTTGCCTGTATGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTG SEQ ID pPR-EcRS-7GTGACGGACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGGCCCGATC NO.: 32 (active site)GCACTCACGTGTGGCTTCGATCCTACCGCTGACAGCTTGCATTTGGGGC SynthetaseATCTTGTTCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGCCACAA poly-GCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAG nucleotideCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAATCTGCTGCAGGGTTATTCGGCTGCCTGTCTTAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCG TCGTCTGCATCAGAATCAGGTGSEQ ID pPR-EcRS-8 CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGANO.: 33 (active site) CTGGCGCAAGGCCCGATCGCACTCGTTTGTGGCTTCGATCCTACCGCTGSynthetase ACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC poly-CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT nucleotideCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATTCGATGGCCTGTACGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTG SEQ ID pPR-EcRS-9CGGGGGCTGGTANCCCAAGTGACGGACGGGGAAGCGTTAGCAGAGCGA NO.: 34 (active site)CTGGCGCAAGGCCCGATCGCACTCAGTTGTGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC poly-CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT nucleotideCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATCTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATAGTTTTGCCTGTCTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTG SEQ ID pPR-EcRS-10CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 35 (active site)CTGGCGCAAGGCCCGATCGCACTCACGTGTGGCTTCGATCCTACCGCTG SynthetaseACACCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC poly-CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT nucleotideCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATACGTTTGCCTGTACTAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTG SEQ ID p-iodoPheRS-1MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALVCGFDPTADSLHLGH NO.: 36 SynthetaseLVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWV Amino acidDKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSYACLNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID p-iodoPheRS-2 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALICGFDPTADSLHLGHNO.: 37  Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSMACLNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID p-iodoPheRS-3 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALVCGFDPTADSLHLGHNO.: 38 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSMACANKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTEMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID OMeTyrRS-1 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALVCGFDPTADSLHLGHNO.: 39 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSMACLNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID OMeTyrRS-2 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALTCGFDPTADSLHLGHNO.: 40 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYTMACLNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID OMeTyrRS-3 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALTCGFDPTADSLHLGHNO.: 41 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYTYACLNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID OMeTyrRS-4 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALLCGFDPTADSLHLGHNO.: 42 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSMACSNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID OMeTyrRS-5 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALLCGFDPTADSLHLGHNO.: 43 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSMACANKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID OMeTyrRS-6 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALTCGEDPTADSLHLGHNO.: 44 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYRMACLNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID p- MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALICGFDPTADSLHLGH NO.: 45acetylPheRS-1 LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVSynthetase DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM Amino acidINKEAVKQRLNREDQGISFTEFSYNLLQGYGMACANKQYGVVLQIGGSDQ (aa)WGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID p- MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALGCGFDPTADSLHLGH NO.: 46benzoylPheRS- LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWV1 Synthetase DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMAmino acid INKEAVKQRLNREDQGISFTEFSYNLLQGYGFACANKQYGVVLQIGGSDQ (aa)WGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKEGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVRGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID p- MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALGCGFDPTADSLHLGH NO.: 47benzoylPheRS- LVPLLCLKRFQQAGRKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWV2 Synthetase DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMAmino acid INKEAVKQRLNREDQGISFTEFSYNLLQGYGYACMNKQYGVVLQIGGSDQ (aa)WGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID p-azidoPheRS- MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALLCGEDPTADSLHLGHNO.: 48 1 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSMACANKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID p-azidoPheRS- MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALVCGFDPTADSLHLGHNO.: 49 2 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSAACANKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID p-azidoPheRS- MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALLCGFDPTADSLHLGHNO.: 50 3 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSAACANKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFTTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID p-azidoPheRS- MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALVCGFDPTADSLHLGHNO.: 51  4 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSAACVNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID p-azidoPheRS- MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALICGFDPTADSLHLGHNO.: 52 5 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANDYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYNFACVNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID p-azidoPheRS- MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALTCGFDPTADSLHLGHNO.: 53 6 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSAACLNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID pPR-EcRS-1 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALGCGFDPTADSLHLGHNO.: 54 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSMACLNKQYGVVLQIGGSDQ p-WGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTS propargyloxy-PYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVL phenylalanineAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEM synthetaseEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID pPR-EcRS-2 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALTCGFDPTADSLHLGHNO.: 55 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSAACLNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID pPR-EcRS-3 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALSCGFDPTADSLHLGHNO.: 56 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYTMACVNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID pPR-EcRS-4 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALACGFDPTADSLHLGHNO.: 57 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSYACLNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID pPR-EcRS-5 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALACGFDPTADSLHLGHNO.: 58 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYTMACCNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTEMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID pPR-EcRS-6 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALTCGFDPTADSLHLGHNO.: 59 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYTFACMNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID pPR-EcRS-7 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALTCGFDPTADSLHLGHNO.: 60 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSVACLNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID pPR-EcRS-8 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALVCGFDPTADSLHLGHNO.: 61 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSMACTNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID pPR-EcRS-9 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALSCGFDPTADSLHLGHNO.: 62 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNELQGYSFACLNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID pPR-EeRS-10 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALTCGFDPTADSLHLGHNO.: 63 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNILLQGYTFACTNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTEMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID tRNA/Tyr AGCTTCCCGATAAGGGAGCAGGCCAGTAAAAAGCATTACCCCGTGGTG NO.: 64poly- GGGTTCCCGAGCGGCCAAAGGGAGCAGACTCTAAATCTGCCGTCATCG nucleotideACCTCGAAGGTTCGAATCCTTCCCCCACCACCA SEQ ID tRNA/TyrAGCUUCCCGAUAAGGGAGCAGGCCAGUAAAAAGCAUUACCCCGUGGU NO.: 65GGGGUUCCCGAGCGGCCAAAGGGAGCAGACUCUAAAUCUGCCGUCAUCGACCUCGAAGGUUCGAAUCCUUCCCCCACCACCA SEQ ID Amber5′-ATGAAGTAGCTGTCTTCTATCGAACAAGCATGCG-3′ NO.: 66 Mutants L3TAG SEQ IDAmber 5′-CGAACAAGCATGCGATTAGTGCCGACTTAAAAAG-3′ NO.: 67 Mutants I13TAGSEQ ID Amber 5′-CGCTACTCTCCCAAATAGAAAAGGTCTCCGCTG-3′ NO.: 68 MutantsT44TAG SEQ ID Amber 5′-CTGGAACAGCTATAGCTACTGATTTTTCCTCG-3′ NO.: 69Mutants F68TAG SEQ ID Amber 5′-GCCGTCACAGATTAGTTGGCTTCAGTGGAGACTG-3′NO.: 70 Mutants R110TAG SEQ ID Amber5′-GATTGGCTTCATAGGAGACTGATATGCTCTAAC-3′ NO.: 71 Mutants V114TAG SEQ IDAmber 5′-GCCTCTATAGTTGAGACAGCATAGAATAATGCG-3′ NO. :72 Mutants T121TAGSEQ ID Amber 5′-GAGACAGCATAGATAGAGTGCGACATCATCATCGG-3′ NO.: 73 MutantsI127TAG SEQ ID Amber 5′-GAATAAGTGCGACATAGTCATCGGAAGAGAGTAGTAG-3′ NO.: 74Mutants S131TAG SEQ ID Amber 5′-GGTCAAAGACAGTTGTAGGTATCGATTGACTCGGC-3′NO.: 75 Mutants T145TAG SEQ ID Permissive5′-CGCTACTCTCCCCAAATTTAAAAGGTCTCCGCTG-3′ NO.: 76 Site Mutants T44FSEQ ID Permissive 5′-CGCTACTCTCCCCAAATATAAAAGGTCTCCGCTG-3′ NO.: 77Site Mutants T44Y SEQ ID Permissive5′-CGCTACTCTCCCCAAATGGAAAAGGTCTCCGCTG-3′ NO.: 78 Site Mutants T44WSEQ ID Permissive 5′-CGCTACTCTCCCCAAAGATAAAAGGTCTCCGCTG-3′ NO.: 79Site Mutants T44D SEQ ID Permissive5′-CGCTACTCTCCCCAAAAAAAAAAGGTCTCCGCTG-3′ NO.: 80 Site Mutants T44KSEQ ID Permissive 5′-GCCGTCACAGATTTTTTGGCTTCAGTGGAGACTG-3′ NO.: 81Site Mutants R110F SEQ ID Permissive5′-GCCOTCACAGATTATTTGGCTTCAGTGGAGACTG-3′ NO.: 82 Site Mutants R110YSEQ ID Permissive 5′-GCCGTCACAGATTGGTTGGCTTCAGTGGAGACTG-3′ NO.: 83Site Mutants R110W SEQ ID Permissive5′-GCCGTCACAGATGATTTGGCTTCAGTGGAGACTG-3′ NO.: 84 Site Mutants R110DSEQ ID Permissive 5′-GCCGTCACAGATAAATTGGCTTCAGTGGAGACTG-3′ NO.: 85Site Mutants R110K SEQ ID p-MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALICGFDPTADSLHLGH NO.: 86 acetylPheRS-LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWV 1 SynthetaseDKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM Amino acidINKEAVKQRLNREGQGISFTEFSYNLLQGYGMACANKQYGVVLQIGGSDQ (aa)^(a)WGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADEMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLERRGKKNYCLICWKSEQ ID Hybrid tRNA GGUGGGGUAGCGAAGUGGCUAAACGCGGCGGACUCUAAAUCCGCUCCNO: 87 CUUUGGGUUCGGCGGTUCGAAUCCGUCCCCCUCCACCA SEQ ID cDNA forGGTGGGGTAGCGAAGTGGCTAAACGCGGCGGACTCTAAATCCGCTCCC NO: 88 Hybrid tRNATTTGGGTTCGGCGGTTCGAATCCGTCCCCCA SEQ ID amber-GGATTACGCATGCTCAGTGCAATCTTCGGTTGCCTGGACTAGCGCTCCG NO: 89 suppressingGTTTTTCTGTGCTGAACCTCAGGGGACGCCGACACACGTACACGTC tRNA expression insertSEQ ID 3′ flanking GACAAGTGCGGTTTTTTTCTCCAGCTCCCGATGACTTATGGC NO: 90sequence of human tRNA^(Tyr) SEQ ID FTam 73:GTACGAATTCCCGAGATCTGGATTACGCATGCTCAGTGCAATCTTCGGT NO: 91 forward TGCCTGGACTAGCGCTCCGGTTTTTCTGTGC primer SEQ ID FTam 115:AGTCCGCCGCGTTTAGCCACTTCGCTACCCCACCGACGTGTACGTGTGT NO: 92 reverse CGGCGTCCCCTGAGGTTCAGCACAGAAAAACCGGAGCGC primer SEQ ID FTam116:GTGGCTAAACGCGGCGGACTCTAAATCCGCTCCCTTTGGGTTCGGCGGT NO: 93 forward  TCGAATCCGTCCCCCACCAGACAAGTG primer SEQ ID FTam117:GATGCAAGCTTGATGGATCCGCCATAAGTCATCGGGAGCTGGAGAAAA NO: 94 reverse  AAACCGCACTTGTCTGGTGGGGGACGG primer A Box TRGCNNAGY Sequence SEQ ID B BoxGGTTCGANTCC NO: 95 Sequence ^(a)These clones also contain a Asp165Glymutation

What is claimed is:
 1. A method of producing in a vertebrate cell atleast one protein of interest comprising at least one non-natural aminoacid, the method comprising: growing, in an appropriate medium, avertebrate cell that comprises a nucleic acid that comprises at leastone selector codon and encodes the protein of interest expressed by thevertebrate cell; wherein the medium comprises an unnatural amino acidand the vertebrate cell comprises: a tRNA having the nucleotide sequenceset forth in SEQ ID NO: 88 that functions in the cell and recognizes theselector codon; and an orthogonal aminoacyl tRNA synthetase (O-RS) thatpreferentially aminoacylates the O-tRNA with the unnatural amino acid;expressing said protein of interest.
 2. The method of claim 1, whereinthe cell has been stably transfected to comprise the O-tRNA and O-RS. 3.The method of claim 1, wherein the cell has been transiently transfectedto comprise the O-tRNA and O-RS.
 4. The method of claim 1, wherein thecell has been stably transfected to comprise the O-tRNA or O-RS andtransiently transfected to comprise the O-tRNA or O-RS, such that thecell still comprises both a t-RNA and an O-RS.
 5. The method of claim 1wherein the unnatural amino acid is selected from the group consistingof: a p-acetyl-L-phenylalanine, a p-iodo-L-phenylalanine, anO-methyl-L-tyrosine, a p-propargyloxyphenylalanine, anL-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, anO-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, atri-O-acetyl-GlcNAcβ-serine, an L-Dopa, a fluorinated phenylalanine, anisopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine,a phosphonoserine, a phosphonotyrosine, a p-bromophenylalanine, ap-amino-L-phenylalanine, an isopropyl-L-phenylalanine, an unnaturalanalogue of a tyrosine amino acid; an unnatural analogue of a glutamineamino acid; an unnatural analogue of a phenylalanine amino acid; anunnatural analogue of a serine amino acid; an unnatural analogue of athreonine amino acid; an alkyl, aryl, acyl, azido, cyano, halo,hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl,seleno, ester, thioacid, borate, boronate, phospho, phosphono,phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, oramino substituted amino acid, or any combination thereof; an amino acidwith a photoactivatable cross-linker; a spin-labeled amino acid; afluorescent amino acid; a metal binding amino acid; a metal-containingamino acid; a radioactive amino acid; a photocaged and/orphotoisomerizable amino acid; a biotin or biotin-analogue containingamino acid; a keto containing amino acid; an amino acid comprisingpolyethylene glycol or polyether, a heavy atom substituted amino acid; achemically cleavable or photocleavable amino acid; an amino acid with anelongated side chain; an amino acid containing a toxic group; a sugarsubstituted amino acid; a carbon-linked sugar-containing amino acid; aredox-active amino acid; an α-hydroxy containing acid; an amino thioacid; an α,α disubstituted amino acid; a β-amino acid; a cyclic aminoacid other than proline or histidine, and an aromatic amino acid otherthan phenylalanine, tyrosine or tryptophan.
 6. The method of claim 2,wherein the protein comprises a therapeutic protein, a diagnosticprotein, an industrial enzyme, or portion thereof.
 7. The method ofclaim 2, wherein the protein comprises a protein or a portion of aprotein selected from the group consisting of: a cytokine, a growthfactor, a growth factor receptor, an interferon, an interleukin, aninflammatory molecule, an oncogene product, a peptide hormone, a signaltransduction molecule, a steroid hormone receptor, erythropoietin (EPO),insulin, human growth hormone, an Alpha-1 antitrypsin, an Angiostatin,an Antihemolytic factor, an antibody, an Apolipoprotein, an Apoprotein,an Atrial natriuretic factor, an Atrial natriuretic polypeptide, anAtrial peptide, a C-X-C chemokine, T39765, NAP-2, ENA-78, a Gro-a, aGro-b, a Gro-c, an IP-10, a GCP-2, an NAP-4, an SDF-1, a PF4, a MIG, aCalcitonin, a c-kit ligand, a CC chemokine, a Monocyte chemoattractantprotein-1, a Monocyte chemoattractant protein-2, a Monocytechemoattractant protein-3, a Monocyte inflammatory protein-1 alpha, aMonocyte inflammatory protein-1 beta, RANTES, I309, R83915, R91733,HCC1, T58847, D31065, T64262, a CD40, a CD40 ligand, a C-kit Ligand, aCollagen, a Colony stimulating factor (CSF), a Complement factor 5a, aComplement inhibitor, a Complement receptor 1, DHFR, an epithelialNeutrophil Activating Peptide-78, a GROα/MGSA, a GROβ, a GROγ a MIP-1α,a MIP-1δ, a MCP-1, an Epidermal Growth Factor (EGF), an epithelialNeutrophil Activating Peptide, an Exfoliating toxin, a Factor IX, aFactor VII, a Factor VIII, a Factor X, a Fibroblast Growth Factor (FGF),a Fibrinogen, a Fibronectin, a G-CSF, a GM-CSF, a Glucocerebrosidase, aGonadotropin, a Hedgehog protein, a Hemoglobin, a Hepatocyte GrowthFactor (HGF), a Hirudin, a Human serum albumin, an ICAM-1, an ICAM-1receptor, an LFA-1, an LFA-1 receptor, an Insulin-like Growth Factor(IGF), an IGF-I, an IGF-II, an IFN-α, an IFN-β, an IFN-γ, IL-1, an IL-2,an IL-3, an IL-4, an IL-5, an IL-6, an IL-7, an IL-8, an IL-9, an IL-10,an IL-11, an IL-12, a Keratinocyte Growth Factor (KGF), a Lactoferrin, aleukemia inhibitory factor, a Luciferase, a Neurturin, a Neutrophilinhibitory factor (NIF), an oncostatin M, an Osteogenic protein, aParathyroid hormone, a PD-ECSF, a PDGF, a Pleiotropin, a Protein A, aProtein G, a Pyrogenic exotoxins A, B, or C, a Relaxin, a Renin, an SCF,a Soluble complement receptor I, a Soluble I-CAM 1, a Solubleinterleukin receptor, a Soluble TNF receptor, a Somatomedin, aSomatostatin, a Somatotropin, a Streptokinase, a Superantigen, aStaphylococcal enterotoxins, an SEA, an SEB, an SEC1, an SEC2, an SEC3,an SED, an SEE, a Superoxide dismutase (SOD), a Toxic shock syndrometoxin, a Thymosin alpha 1, a Tissue plasminogen activator, a tumorgrowth factor (TGF), a TGF-α, a TGF-β, a Tumor Necrosis Factor, a TumorNecrosis Factor alpha, a Tumor necrosis factor beta, a Tumor necrosisfactor receptor (TNFR), a VLA-4 protein, a VCAM-1 protein, a VascularEndothelial Growth Factor (VEGEF), a Urokinase, a Mos, a Ras, a Raf aMet; a p53, a Tat, a Fos, a Myc, a Jun, a Myb, a Rel, an estrogenreceptor, a progesterone receptor, a testosterone receptor, analdosterone receptor, an LDL receptor, a SCF/c-Kit, a CD40L/CD40, aVLA-4/VCAM-1, an ICAM-1/LFA-1, a hyalurin/CD44, and a corticosterone.